Milk yield (MY), composition, and fatty acid profiles of purebred Holstein (HO) cows, Brown Swiss (BS), and their F crossbreds (HS) were compared under subtropical climate in Egypt. Pure HO had significantly greater 305-MY, total-MY, and daily-MY (p < 0.05) than pure BS and crossbred HS. Furthermore, HO and HS had significantly higher peak-MY (44.2 and 43.3 kg, respectively) than BS (36.1 kg). In comparison with HO, BS had significantly higher milk fat, protein, total solids, and solid-not-fat % (p < 0.05); however, no significant differences were observed between BS and HS for milk fat %. The milk fat of BS had higher concentrations of saturated (C17:0 and C18:0) fatty acids (FA) than that of the HO and HS (p = 0.001 and 0.008, respectively). Content of C4:0, C6:0, C8:0, C12:0, and C15.0 FA did not differ between genotypes. Milk from HO and BS had significantly higher concentrations of unsaturated (C20:1 and C20:5) FA than that from HS (p < 0.05). C14:1 (myristoleic), C16:1 (palmitoleic), and C18:2 (linoleic) FA contents were similar for all genotypes. In conclusion, BS surpassed HO for all milk composition traits and some FA components; however, HS had comparable milk fat percentage with BS. There appears to be an opportunity to modify the concentration of certain FA by breeding.