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The qualities of food, mainly of animal origin, have always been of concern to consumers. It is known that the composition of animals' diets can influence the composition of the foods produced, such as milk. Thus, the objective of this study was to evaluate diets with cactus pear in combination with different forages for F1 Holstein/Zebu cows on the chemical composition and fatty acid profile of milk. Two simultaneous 4 × 4 Latin squares were used in the experimental design. Four experimental diets were used: Diet 1, sorghum silage as the only roughage; Diet 2, sorghum silage combined with cactus pear in a proportion of 50% of the roughage (dry matter basis); Diet 3, elephant grass (Pennisetum purpureum cv. Roxo) as the only roughage; and Diet 4, elephant grass combined with cactus pear in a proportion of 50% of the roughage. The roughage:concentrate ratio was 75:25. The milk urea nitrogen was higher (16.08 mg/dL) in the milk of cows fed the diet with only elephant grass than that of milk from the other diet groups. The other analyzed variables of the chemical composition of milk were not influenced by the diets. However, there was a change in the sum of the saturated and monounsaturated fatty acids. The content of C16:0 fatty acid was higher and that of C18:0 was lower in the milk of cows fed the cactus pear diets than in that of cows fed the other diets. Higher levels of oleic acid were observed in the milk of cows fed with diets containing elephant grass than those in the milk of the cows in the sorghum forage groups. Conjugated linoleic acid (CLA) was higher in the milk of cows fed cactus pear than in that of the other cows. The combination of cactus pear with elephant grass or sorghum silage in the diet did not alter the chemical composition of milk. However, it influenced the fatty acid profile.