Two novel single nucleotide polymorphisms (SNPs) were found in the bovine β4-defensin gene intron – A/C transversion at position 1674 and C/T transition at position 1877 – both recognized with BsrI endonuclease. Observed frequencies of the A1674C AA, AC, CC genotypes were 0.65, 0.32 and 0.03, respectively, while those of alleles A and C – 0.81 and 0.19. For the C1877T polymorphism in the Polish-Friesian (PF) population studied only the CC homozygotes (frequency 0.94) and CT heterozygotes (frequency 0.06) were found. Associations were evaluated between the A1687C polymorphism and milk production traits of cows. The data set comprised 8814 records of daily milk, fat, protein and ECM yields, fat, protein, lactose and dry matter contents, and somatic cell count (SCC) of milk of 352 cows. Moreover, 897 records were used of milk fat and milk protein yield and content achiewed in the whole and the standard 305-day lactation, as well as estimates of breeding values of these traits in 352 animals from official recording of milk performance (National Breeding Programme). In the whole lactation the β4-defensin gene A1674C polymorphism was fund significantly related to protein yield while in the standard lactation to fat and protein contents.Moreover, the polymorphism was found related to the breeding value for protein yield, and for FAT and protein content.