In 2003, 2004 and 2005 milk fat composition was determined three times in 55 dairy cows of Czech Pied cattle housed in a byre with stanchions and fed under conditions usual on Czech production farms. Fatty acids were determined by a gas chromatographic method, 26 acids out of the total 37 acids observed in chromatograms were identified. The highest proportions were observed for palmitic acid (29.25 ± 2.98%), oleic acid (24.47 ± 3.27%), myristic acid (12.14 ± 1.80%) and stearic acid (8.91 ± 2.44%). The proportions of saturated, unsaturated and monounsaturated fatty acids were 64.71 ± 4.18, 31.96 ± 4.20 and 27.45 ± 3.42% of total acids, respectively. The total proportion of nutritionally undesirable lauric, myristic and palmitic acid was 45.26 ± 4.77%, while that of the desirable group of polyunsaturated fatty acids was 4.51 ± 1.09%. The observed relatively wide ranges of the individual groups of fatty acids indicate that it is possible to improve the milk fat composition in Czech Pied cows.