Two identical experimental protocols were followed at 200 and 3,600 m above sea level (a.s.l.) determining the changes of the milk fatty acid (FA) profile of Brown Swiss (BS) and indigenous Peruvian Criollo cows (CR) as a response to diets which were designed to cover the variation in feed quality caused by season. At each site (altitude), six BS and six CR cows, adapted to > 3,500 m a.s.l., were fed three dietary treatments (DS, dry-season forage; RS rainy-season forage; OC, diet optimised to meet the cow's requirements) in a 2 x 2 x 3-factorial arrangement. Intakes of FA and milk yield increased from diet DS (low quality diet) to RS and OC (high quality diet) for both cow types. Milk fat proportions of conjugated linoleic acid (CLA), C18:3 c9,c12,c15, total n-3 and polyunsaturated FA (PUFA) were highest (p < 0.05) with diet OC and higher in the lowlands than in the highlands. Low intakes of diet DS obviously resulted in a ruminal energy deficiency and body lipid mobilisation. The ruminal energy deficiency with diet DS was especially pronounced in BS, apparently reducing biohydrogenation rate and leading to lower proportions of C18:0 and higher proportions of C18:3 c9,c12,c15 in milk fat (p < 0.05). Especially C18:3 c9,c12,c15 intake did not concur with its proportion in milk fat, suggesting a strong dependence on energy status. Milk yield and FA excretion with milk were higher for BS than for CR (p < 0.05) with all three diets although milk fat content was lower (p < 0.05) for BS than CR. Milk fat of BS was richer in CLA and PUFA than milk fat of CR (p < 0.05). The desaturase indices for 18 FA were also higher for BS than CR (p < 0.05), suggesting a slightly higher delta9-desaturase activity for BS, especially with diet DS. Milk fat content was generally higher at the high altitude than at the lowland site (p < 0.05), whereas the FA profile was unexpectedly similar across sites. Various interactions were found among diet type, cow type and altitude (site) indicating that a combination of these factors contributes to the characteristic FA profile of the respective milk.