Four years of experimental work were conducted applying PCR-RFLP to detect the SNP polymorphic associations of the prolactin candidate gene (PRL) with milk traits in cattle. A total of 494 lactations for 180 milking cows of Friesian in Elkarda herd (FK) and Sakha herd (FS) and Baladi cows in Elserw herd (LS) were used. The PCR products were digested using NaeI and SmaI restricted enzymes to investigate the molecular associations of the PRL gene genotypes with milk traits. The highest expected numbers of alleles (NE) in CC, CD and DD genotypes of the PRL gene were recorded in the DD genotype in both Friesian and Baladi herds, while the lowest expected numbers were recorded in CC genotype in all herds. The highest NE in GG, GT and TT genotype was in the TT genotype and the lowest NE was in the GG genotype in all herds. PRL gene was not in Hardy-Weinberg Equilibrium in all herds. The allelic frequency for the D allele was much higher than the frequency of the C allele, while the frequency of the T allele was higher than the frequency of the G allele. The observed and expected values of heterozygosity for the PRL gene were mostly moderate in the three herds studied. The values of polymorphic information content (PIC) were mostly moderate in all herds. The values of fixation index (reduction in heterozygosity due to inbreeding, FIS) were high in the FK herd (0.82), FS herd (0.62) and BS herd (0.94). The molecular associations between PRL gene genotypes and milk traits indicate that the CD genotype of the PRL gene has strong molecular associations with yields of milk, fat and protein relative to CC and DD genotypes. GG genotype was significantly associated with all milk traits relative to GT and TT genotypes.