The objective of this study was to identify DNA markers in the 4 casein genes (CSN1S1, CSN1S2, CSN2, and CSN3) and the 2 major whey protein genes (LALBA and LGB) that show associations with milk protein profile measured by reverse-phase HPLC. Fifty-three single nucleotide polymorphisms (SNP) were genotyped for cows in a unique resource population consisting of purebred Holstein and (Holstein x Jersey) x Holstein crossbred animals. Seven traits were analyzed, including concentrations of α(S)-casein (CN), β-CN, κ-CN, α-lactalbumin, β-lactoglobulin, and 2 additional secondary traits, the total concentration of the above 5 milk proteins and the α(S)-CN to β-CN ratio. A substantial fraction of phenotypic variation could be explained by the additive genetic component for the 7 milk protein composition traits studied. Moreover, several SNP were significantly associated with all examined traits at an experiment-wise error rate of 0.05, except for α-lactalbumin. Importantly, the significant SNP explained a large proportion of the phenotypic variation of milk protein composition. Our findings could be used for selecting animals that produce milk with desired composition or desired processing and manufacturing properties.