3-Nitrooxypropanol (3-NOP) is a promising and approved antimethanogenic feed additive in dairy farming. The objective of this study was to evaluate breed-specific variation in milk fatty acids (FA) following 3-NOP supplementation. Milk FA composition was analyzed from 3 studies using Danish Holstein (DH), Danish Jersey (DJ), and Danish Red (DR) dairy cows respectively, fed control or 3-NOP supplemented diets. The 3-NOP was supplemented at a dose of 60 mg/kg feed DM. Bulk milk samples were analyzed for milk FA by GC flame ionization detection. The 3-NOP supplemented diets increased the proportion of short-chain fatty acids (SCFA) and decreased the proportion of C16:0 in the milk of DH cows. A similar tendency in milk FA composition induced by 3-NOP was observed in DJ cows; however, the extent of these changes was less pronounced, and no changes were observed in milk FA from DR cows. Indirect comparison among the breeds showed a higher proportion of SCFA in milk from DJ cows, a higher proportion of medium-chain FA in DR cows and a higher proportion of long-chain FA in DH cows. The effect of 3-NOP on milk FA composition appears to vary between breeds; however, this variation may also be influenced by differences in feeding regimens between breeds, making further studies worthwhile to better understand these interactions.