The aim of this study was to estimate allelic and genotypic frequencies of five DNA markers that are positional and functional candidates for milk production traits in Czech Fleckvieh cattle. In addition, we evaluated the association of these markers with milk production traits and breeding values for milk production traits and also estimated linkage disequilibrium (LD) between two markers within the prolactin (PRL) gene. As part of this study, 505 Czech Fleckvieh cows were genotyped. The markers in proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), secreted phosphoprotein (SPP1), cytochrome P450 family 11 subfamily B hydroxylase (CYP11B1), and the two polymorphisms in the prolactin gene (PRL) showed evidence of segregation in our study. The PPARGC1A polymorphism was associated with milk yield, milk fat and protein traits. The polymorphism in SPP1 was significantly associated with milk protein percentage. The CYP11B1 polymorphism showed positive associations with milk composition traits and breeding values for milk yield, milk fat, and protein traits. Both polymorphisms within the PRL gene were associated with milk yield, milk fat and milk protein yield (individually and grouped). Linkage disequilibrium between the two polymorphisms in PRL was not observed. In conclusion, all markers examined in this study are important markers for milk production traits in Czech Fleckvieh cattle, and both markers within the PRL gene should be evaluated in future research.