Abstract: Seabuckthorn germplasm resources with excellent fruit quality can greatly contribute to the development and utilization of cold seabuckthorn germplasm resources and the breeding of new varieties. The fruit quality analysis and comprehensive evaluation can be effectively utilized to screen the seabuckthorn germplasm resources in recent years. In this study, a series of evaluations was implemented on 22 seabuckthorn germplasm resources with 10 indices using the coefficient of variability, correlation analysis, principal component analysis (PCA), and cluster analysis. A comprehensive score was then ranked to evaluate the seabuckthorn fruit quality. The specific calculation steps were as follows. The first step was the measurement of fruit quality indicators. The measured indexes included 10 indexes of hundred fruit weight, fruit longitudinal diameter, fruit transverse diameter, fruit shape index, soluble solids contents, soluble sugar, titratable acidity, ascorbic acid, solid acid ratio, and Sugar acid ratio. The second step was to conduct coefficient of variation analysis, PCA, Cluster analysis, and the dynamic correlation heatmap analysis. The third step was to construct the fruit comprehensive evaluation model. As such, 22 seabuckthorn germplasm resources fruit quality comprehensive scores were obtained using the improved model. The fruit quality of each resource was also ranked, according to the score. There was the nonuniform external appearance and internal quality of the 22 seabuckthorn germplasm resources. The hundred fruit weight ranged from 21.035 to 78.950 g. The fruit's longitudinal diameter ranged from 0.705 to 1.455 mm. The fruit transverse diameter ranged from 0.710 to 1.005 mm. The fruit shape index ranged from 0.947 to 1.617. The soluble solids contents ranged from 5.775% to 9.650%. The soluble sugar ranged from 0.695% to 3.885%. The titratable acidity ranged from 0.975% to 2.980%. The ascorbic acid ranged from 97.575 to 284.920 mg/100g. The solid acid ratio ranged from 0.311 to 3.769. The sugar-acid ratio ranged from 2.151 to 9.797. The results showed that there was a great difference in the quality traits of seabuckthorn germplasm resources fruit, with the coefficient of variation ranging from 9.339% to 62.528%. The variation coefficients were ranked in descending order: sugar acid ratio (62.528%), soluble sugar (43.988%), solid acid ratio (35.927%), titratable acidity (35.150%), ascorbic acid (32.490%), hundred fruit weight (31.728%), fruit transverse diameter (9.339%), fruit shape index (14.375%), soluble solids contents (13.921%), and fruit longitudinal diameter (18.076%). A larger coefficient of variation was found in the important fruit quality indexes, such as soluble sugar content, titratable acid content, and ascorbic acid content. A strong reference index was offered for parental selection and variety improvement in seabuckthorn breeding. Correlation analysis showed that there were different degrees of correlation among each quality index. Three principal components were extracted by PCA analysis, where the cumulative contribution rate was 86.053%. The first principal component explained 62.593% of trait information, indicating the high correlation with hundred fruit weight, sugar acid ratio, solid acid ratio, fruit longitudinal diameter, and soluble sugar. The second principal component explained 12.597% of trait information, indicating the high correlation with fruit shape index. The third principal component explained 10.863% of trait information with a high correlation to the soluble solid contents. The PCA avoided the subjective influence of artificial evaluation on the comprehensive evaluation of fruit quality, in order to comprehensively and objectively evaluate the fruit quality of seabuckthorn germplasm resources. Furthermore, a comprehensive evaluation model was established for the fruit quality of seabuckthorn germplasm resources, according to the scores of the three principal components and the corresponding weights. The comprehensive scores of each resource were obtained with the descending order of: ‘Shicong’, ‘Ivshca’, ‘Xiangyang’, HS-12, ‘Yousheng’, HS-10, ‘Zeliangnü’, HS-6, ‘Qiji 2’, ‘Chuyi’, ‘Zirianka’, ‘Wulangemu’, HS-4, ‘Shiyou 2’, ‘Ornistaya’, ‘Zhuangyuanhuang’, Meng 1, ‘Shenqiuhong’, ‘Zhiwuyuan’, ‘Suiji 2’, ‘Liaofu 1’, ‘Shiyou 1’. The cluster analysis indicated that the 22 seabuckthorn germplasm resources were divided into three groups in the cluster analysis at a Eudlidean distance of 12. According to the cluster analysis, class I was the best in fruit quality, including ‘Shicong’, ‘Xiaoliushu’, HS-12 and ‘Xiangyang’ with bigger fruit weight, higher soluble solid content, higher soluble sugar content, and lower titrable acid content. HS-4, ‘Chuyi’, HS-6, HS-10, ‘Yousheng’, ‘Zeliangnü’, ‘Qiji 2’, ‘Wulangemu’, ‘Zhuangyuanhuang’, Meng 1, ‘Shiyou 2’, ‘Huoguang’ and ‘Zeliang’ were classified as class II; Class III included ‘Liaofu 1’, ‘Zhiwuyuan’, ‘Shiyou 1’, ‘Suiji 2’, and ‘Shenqiuhong’ with the smaller fruit weight, lower soluble solid content, lower soluble sugar content, and higher titrable acid content. The classification was highly consistent with those of the principal component score. Therefore, ‘Ivshca’, ‘Shicong’, ‘Xiangyang’ and HS-12 can be used as excellent germplasm resources in the cold region. The comprehensive evaluation model reduced the workload of fruit quality data analysis, in order to improve the efficiency of the selection and breeding of new seabuckthorn varieties. Moreover, a more accurate and reliable evaluation was achieved in the seabuckthorn germplasm resources fruit quality. The finding can provide the scientific basis for quality evaluation, the selection and breeding, improvement and planting scheme of new seabuckthorn varieties.