Fatty acid (FA) composition of longissimus dorsi intramuscular fat in Black Yak and Chinese Yellow Cattle were evaluated in 44 Black Yak and 41 Chinese Yellow Cattle of both genders. Interactions of species with gender were observed for total saturated fatty acid (SFA), unsaturated fatty acid (UFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), palmitic acid, stearic acid, oleic acid, linolenic acid, arachidonic acid, EPA, and DHA concentrations, as well as PUFA/SFA ratio in the longissimus dorsi (p<0.05). The SFA percentage was greater in yellow cattle than yak in both genders but the species difference in heifers was greater than in steers (p<0.05). Yak had greater UFA, MUFA and PUFA percentages than yellow cattle in both steers and heifers (p<0.05) but the difference between yak and yellow cattle heifers was greater than yak and yellow cattle steers. The percentages of inolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid; and PUFA/SFA were greater in yak than yellow cattle in both steers and heifers (p<0.05). In addition, the ratio of n-6/n-3 PUFA in yak was lesser than in yellow cattle (p<0.05). These results indicated that FA composition generally differed between yak and yellow cattle but the differences were not the same in heifers as compared to steers. Results also suggested that species differences in FA composition tended to favor Black Yak over Chinese Yellow Cattle, indicating that the longissimus dorsi of Black Yak may have a higher nutritive value than that of Chinese Yellow Cattle and potential for development as a desirable natural product.