Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene which is associated with development and growth in livestock. The present study aimed to investigate the polymorphisms of IGFBP-3 gene in Holstein Friesian (HF) and Anatolian Black (AB) cattle breeds. BsuRI (GG↓CC) restriction enzyme was used to detect of IGFBP-3 gene polymorphism. Although Anatolian Black breed was monomorphic (AA genotype), three genotypes (AA, AB and BB) were detected in Holstein Friesian breed by digestion of PCR products with BsuRI . The A and B allele frequencies were 0.57 and 0.43, respectively, in Holstein Friesian breed, while A allele frequency was 1.00 in Anatolian Black breed. AA, AB and BB genotype frequencies were 0.32, 0.50 and 0.18, respectively in the Holstein Friesian breed. All three possible genotypes were detected in Holstein Friesian breed, significant deviation from Hardy-Weinberg equilibrium (P<0.05) was observed in terms of genotype distributions.