Dairy products are a significant contributor to the fat intake of the American diet and contain several bioactive fatty acids (FA) that possess health benefits. The objective of this study was to compare the content and profile of bioactive FA in milk between Holstein (HO), Jersey (JE), and F₁ HO × JE crossbreds (CB). Twenty-two primiparous cows (n = 7 HO, n = 7 CB, n = 8 JE) were followed across a lactation. All cows were fed a consistent TMR at a 70:30 forage to concentrate ratio. Sample periods (SP) were defined as 3 d in milk (DIM), 93 DIM, 183 DIM, and 273 DIM. Milk sample and data collection occurred on days −2 to 2 relative to the SP. Data were analyzed using a repeated measures general linear mixed model in SAS. HO and CB, had a higher content of n-3 FA in milk fat than JE (0.60 and 0.63 vs. 0.47 g/100g FA; P < 0.01) and a lower n-6/n-3 ratio (3.2 and 3.1 vs. 3.8; P < 0.01). SP had an effect on the n-6/n-3 ratio of milk fat, with the lowest value observed at 3 DIM (2.6; P < 0.01) and the highest at 183 DIM (3.9; P < 0.01). The content of conjugated linoleic acids (CLA) in milk fat was affected by breed and SP. HO and CB had a higher proportion of CLA than JE (0.50 and 0.48 vs. 0.37 g/100g FA; P < 0.01). Overall, CLA content decreased over lactation (0.54, 0.45, 0.40, and 0.40 g/100g FA for 3, 93, 183 and 273 DIM, respectively; P < 0.01). Breed had no effect on content of total branched-chain fatty acids (BCFA) in milk fat, however, breed affected individual BCFA. Content of iso 14:0 in milk fat was lower in HO than in JE (0.10 vs. 0.13 g/100g FA; P < 0.05) and content of iso 17:0 was higher in HO and CB than in JE (0.33 and 0.31 vs. 0.27g/100g FA; P < 0.01). Total BCFA were affected by SP, with 93 and 183 DIM (1.51 and 1.59 g/100g FA) having a lower proportion of BCFA than 3 and 273 DIM (1.71 and 1.74 g/100g FA; P < 0.01). In conclusion, HO and CB exhibited a higher content of bioactive FA in milk than JE. Across a lactation, the greatest content of bioactive FA in milk occurred postpartum.