Fractionation is important for the application of milk fat (MF). In this study, the contents of fatty acid (FA) and triglyceride (TAG) of MF and its fractions distilled by short-path molecular distillation (SPMD) were detected. The results showed that a total of 19 FAs and 109 TAG molecular species were detected. Moreover, the short-chain saturated FA, medium-chain saturated FA, low-molecular-weight TAG, medium-molecular-weight TAG, S 3 (TAG molecular species with three saturated FAs) and L 2 S (TAG molecular species with two long-chain FAs and one short-chain FA) were easily accumulated in the distillate, and the percentage of these components all increased first and then decreased during the whole distilling process. Compared with the distillate, polyunsaturated FA (PUFA) and high-molecular-weight TAG (HMW-TAG, with the carbon numbers: 41-54 and molecular weight: 704-888) were enriched in the residue, and the increasing ratio of PUFA and HMW-TAG was 393.75% and 8.58% respectively. Further analysis showed that the 16:0/4:0/16:0, 16:0/4:0/14:0, 16:0/12:0/4:0, etc. were the discrepant TAG molecular species during SPMD. Therefore, these results demonstrated that different fractions of MF could be obtained by adjusting the fractionation temperature, and it also would provide more important theoretical guidance for regulating MF fractionation, enriching the nutritional information of MF fractions.