The aim of this study was to investigate the gene and genotype distributions of some mutations in the aromatase cytochrome P450 (CYP19), estrogen receptor α (ERα), and progesterone receptor (PGR) genes in fertile and subfertile Holstein-Friesian heifers using the PCR-RFLP method and comparing the distributions between groups. A total of 106 heifers were included the study, and the heifers that became pregnant after the first artificial insemination (n=51) were used as a fertile group. Heifers (n=55) with equal and more than 3 AIs were accepted as a subfertile heifers. Blood samples from all of the heifers were obtained for DNA isolation. While two alleles and three genotypes were found at the PGR and ERα loci, two alleles and two genotypes were detected at the CYP19 locus. The A allele and AA genotype, G allele and GG genotype, and C allele and CT genotype were found to be predominant in CYP19, ERα and PGR, respectively. According to the chi-square test (χ2), two of the groups investigated were in Hardy-Weinberg equilibrium for all gene loci. There were no differences detected in allele or genotype frequencies between the fertile and subfertile heifers. Keywords