Dairy products contain bioactive fatty acids (FA) and are a unique dietary source of an emerging class of bioactive FA, branched-chain fatty acids (BCFA). The objective of this study was to compare the content and profile of bioactive FA in milk, with emphasis on BCFA, among Holstein (HO), Jersey (JE), and first generation HO x JE crossbreeds (CB) across a lactation to better understand the impact of these factors on FA of interest to human health. Twenty-two primiparous cows (n = 7 HO, n = 7 CB, n = 8 JE) were followed across a lactation. All cows were fed a consistent total mixed ration (TMR) at a 70:30 forage to concentrate ratio. Time points were defined as 5 days in milk (DIM), 95 DIM, 185 DIM, and 275 DIM. HO and CB had a higher content of n-3 FA at 5 DIM than JE and a lower n-6:n-3 ratio. Time point had an effect on the n-6:n-3 ratio, with the lowest value observed at 5 DIM and the highest at 185 DIM. The content of vaccenic acid was highest at 5 DIM, yet rumenic acid was unaffected by time point or breed. Total odd and BCFA (OBCFA) were higher in JE than HO and CB at 185 and 275 DIM. Breed affected the content of individual BCFA. The content of iso-14:0 and iso-16:0 in milk was higher in JE than HO and CB from 95 to 275 DIM. Total OBCFA were affected by time point, with the highest content in milk at 275 DIM. In conclusion, HO and CB exhibited a higher content of several bioactive FA in milk than JE. Across a lactation the greatest content of bioactive FA in milk occurred at 5 DIM and OBCFA were highest at 275 DIM.