The impact of polymorphism of the diacylglycerol acyltransferase (DGAT1), butyrophilin (BTN1A1), oxidized low-density lipoprotein receptor (OLR1), and signal transducer and activator of transcription 1 (STAT1) genes on milk production and reproduction traits in 419 Czech Fleckvieh cows was examined using polymerase chain reaction and restriction fragment length polymorphism. The loci DGAT1 and BTN1A1 were observed simultaneously to affect milk production, estimated breeding value of milk production traits, as well as reproduction parameters. Significant differences were found also between genotypes of the STAT1 loci in relation to estimated breeding value of milk production traits. Similar findings in pure dairy breeds suggest that heterogeneous effects of the observed loci can be explained by different genetic backgrounds in various breed populations selected to achieve different commercial goals. Thus, it is necessary to determine variability and influence of a molecular marker in a specific population when considering its inclusion into a breeding programme.