The aim of this research was to study the potential for selection of cows with a higher nutritional quality of milk fat by studying the differences in fatty acid profiles within and across the following breeds: Dual Purpose Belgian Blue, Holstein-Friesian, Jersey, Montbeliarde, and non-Holstein Meuse-Rhine-Yssel type Red and White. Six hundred milk samples from 275 animals were taken from 7 herds. Several types of fatty acids in milk and milk fat were quantified using mid-infrared spectrometry and previously obtained calibration equations. Statistical analyses were made using a mixed linear model with a random animal effect. The variance components were estimated by using REML. Results showed breed differences for the fatty acid profile. The repeatability estimate obtained in the present study may suggest the existence of moderate additive genetic variance for the fatty acid profile within each breed. Results also indicated variation for each analyzed milk component in the whole cow population studied. Genetic improvement of the nutritional quality of milk fat based on fatty acid profiles might be possible, and further research and development are warranted.