The purpose of the study was to analyse changes in the frequency of genotypes and alleles of beta-casein (CASB), kappa-casein (CSN3) and betalactoglobulin (LGB) loci among bulls (used for insemination in 1991–2021) and cows (milked in 2014 and 2021). The analysis included 1812 bulls and 2628 cows of the Polish Holstein-Friesian breed. Genotypes of three milk protein loci were identified using PCR-RFLP/ACRS method (bulls) or the Illumina Bovine MDv2 Chip (cows). For bulls used in insemination between 1991 and 2021, the frequency of the desirable CSN3 allele B and allele A2 at the CASB locus increased by 44% and 8%, respectively. In cows, the frequency of allele B of CSN3 and allele A2 of CASB increased over the past seven years by 11% and 7%, respectively. The frequency of LGB allele B decreased in cows from 47% to 41%. An increase in the frequency of the rarest allele E at the CSN3 locus in bulls was also observed (from 2% to 7%). The number of animals carrying the most desirable combination of CASB and CSN3 genotypes (A2A2 and BB) reached 4.25% and 2.8% in cows and bulls, respectively. The current results showed a slow but marked increase in the desirable alleles at casein gene loci which should improve milk quality and cheese-processing properties. This positive effect was attenuated by the simultaneous decrease in the frequency of LGB allele B.