In the present study, we aimed to investigate the changes in triacylglycerol (TAG) composition as affected by alterations in the cows' diet due to seasonal variations and genetic factors. For this study, 50 milk fat samples in winter and 50 in summer were used from 25 cows with the DGAT1 KK genotype and 25 cows with the DGAT1 AA genotype. The samples were analyzed for milk fat content (%), fat composition, and TAG composition. We found that the content of TAG species CN54 was higher and that of CN34 and CN36 lower in summer than in winter. This seasonal variation in TAG profile was related to seasonal changes in the fatty acids C14:0, C16:0, C18:0, C18:1 cis-9, total unsaturated fatty acids, and total long-chain fatty acids, most likely resulting from dietary differences between seasons. Furthermore, we quantified the effect of DGAT1 K232A polymorphism on TAG profile and detected a significant effect on TAG species CN36, with higher values for the DGAT1 KK genotype. When adjusting for differences in fat content, we found no significant effects of the DGAT1 K232A polymorphism on TAG profile. We detected a significant interaction between DGAT1 K232A polymorphism and season for TAG species CN42 and CN52; in summer, the KK genotype was associated with higher levels for CN42 than the AA genotype, whereas in winter, the difference between the genotypes was small. For CN52, in summer the AA genotype was associated with higher levels than the KK genotype. In winter, the difference between the genotypes was also small. We show that, regardless of preference for DGAT1 genotype (AA or KK) and depending on the availability of FA according to season, UFA (C18:1 cis-9), short-chain FA (C6:0 and C10:0), and medium-chain FA might be esterified on the glycerol backbone of the TAG, keeping the structure characteristics of each TAG species. To our knowledge, this is the first report on the interaction effect of DGAT1 K232A polymorphism and season on the TAG composition in milk fat.