The aim of our study was to estimate effects of polymorphisms in the ATP-binding cassette G2 (ABCG2), fatty acid synthase (FASN), oxidized low-density lipoprotein receptor 1 (OLR1), peroxysome proliferator-activated receptor-γ coactivator-1α (PPARGC1A), prolactin (PRL) and signal transducer and activator of transcription 5A (STAT5A) genes on milk production traits and detailed milk-fat composition. Milk-fat composition phenotypes were available for 1905 Dutch Holstein–Friesian cows. First, the presence of each SNP in the Dutch Holstein–Friesian population was evaluated by direct sequencing of the PCR product surrounding the SNP in 22 proven Dutch Holstein–Friesian bulls. The ABCG2 SNP did not segregate in the bull population. Second, we genotyped the cows for the FASNg.16024G>A, FASNg.17924A>G, OLR1g.8232C>A, PPARGC1Ac.1790+514G>A, PPARGC1Ac.1892+19G>A, PRLg.8398G>A and STAT5Ag.9501G>A polymorphisms, and estimated genotype effects on milk production traits and milk-fat composition. FASNg.17924A>G and OLRg.8232C>A had a significant effect (P < 0.05) on milk-fat percentage. However, we were not able to confirm results reported in the literature that showed effects of all evaluated polymorphisms on milk-fat percentage or milk-fat yield. All polymorphisms showed significant effects (P < 0.05) on milk-fat composition. The polymorphisms in FASN and STAT5A, which had an effect on C14:0 and were located on chromosome 19, could not fully explain the quantitative trait locus for C14:0 that was previously detected on chromosome 19 in a genome-wide scan using linkage analysis.