Abstract: Rapid identification of alfalfa seedling salt tolerance has important signification for salt-tolerant germplasms screening and salt-tolerant new varieties breeding. Proline and MDA are two important biochemical indicators of plant salt tolerance. Near infrared spectroscopy calibrations of proline and MDA in the seedling of 40 different alfalfa varieties were studied by means of partial least-squares (PLS) regression in the paper. The calibrations for fresh and dried samples of alfalfa seedling were analyzed by near infrared diffuse reflectance spectroscopy (NIDRS). Good calibrations of NIDRS were found for proline and MDA, the correlation coefficients of calibration (R2) and correlation coefficients of validation (r2) were all greater than 0.85, and the ratio of performance to standard deviation (RPD)value of proline and MDA for fresh and dried samples were 1.72, 2.04 and 2.38, 3.97. The results of flesh samples and dried samples were not evident (P>0.05). The verification results of seedling salt tolerance in 12 alfalfa cultivars show that the proline and MDA model of near infrared spectroscopy can rapidly and accurately be used to identify seedling salt tolerance of alfalfa.