Abstract The objectives of this study were to evaluate the impact of vaccine program and progression of grazing season on beef cow milk fatty acid composition. In 2015, Fifty-one Angus-crossed beef heifers were randomly assigned to receive a modified-live viral vaccine (MLV; Bovishield Gold 5 FP) or killed viral vaccine (KV; Cattlemaster Gold 5 FP) against BVDV beginning at 3 months of age and for the duration of their lifespan in the herd. In 2019, 2020, and 2021, milk samples were collected on days 1, 63, and 205 of lactation. Milk samples were subjected to fatty acid analysis using Gas Chromatography. Data were analyzed as a completely randomized design with repeated measures using SAS. Milk concentrations of 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, or total fatty acids when expressed as mg/g were not affected by vaccine treatment (P ≥ 0.58). However, milk 16:0 concentrations decreased linearly (P < 0.0001) as the grazing season progressed from day 1 to day 205 of lactation. This was despite a linear increase (P = 0.05) in forage concentrations of 16:0. Milk concentrations of 18:0 decreased linearly throughout lactation (P = 0.002). Likewise, milk concentrations of 18:1n-9 decreased throughout lactation (P < 0.0001). Milk concentrations of 18:2n-6 declined linearly (P < 0.0001) as the grazing season progressed into the fall weaning period. Interestingly, milk concentration of 18:3n-3 did not change (P = 0.62) with time. Biohydrogenation intermediates were reduced in concentration and did not vary across grazing season (P ≥ 0.06) Total fatty acids decreased throughout lactation (P < 0.0001). Total fatty acid content of forage did increase linearly (P = 0.02), which would suggest milk production (volume) and de novo milk fatty acid synthesis altered milk composition more so than forage fatty acid composition. In conclusion, vaccine treatment did not affect milk composition. However, grazing season progression resulted in decreased milk fatty acid concentrations from day 1 to day 205 of lactation.