Supernumerary teats (SNT) are a prevalent and intricate polygenic trait that is frequently observed at birth in sheep. These additional teats have been linked to a heritable trait associated with maternal capability, especially in ewes with high fertility. Investigating the genomic basis of these characteristics is an ongoing endeavor and poses an intriguing research inquiry with implications for both biological understanding and practical applications. In order to delve into this scientific enigma, a specific cohort of 20 local fat-tailed Ghezel ewes, each possessing four functional teats, was carefully chosen to form the case group. In parallel, a control group comprising 25 healthy ewes with the standard two normal teats was established for com-parative analysis. Blood samples were meticulously collected from both groups of Ghezel ewes demonstrat-ing distinct phenotypic expressions, specifically those with either two or four functional teats. Following the sample collection, the genomic DNA extraction process was carried out using a commercial DNA purifica-tion kit provided by Qiagen company, known for its high-quality DNA extraction products. We conducted an examination of four PCR-RFLP markers on four candidate genes (IGF1, Leptin, Calpastatin, and FBP30). Through our research, we unveiled the allele distribution and genotype frequency of these potential genes in both the case and control groups of sheep. Our findings demonstrated notable disparities in the genotype and gene frequencies between the case and control groups for each candidate gene. Furthermore, we calculated the odds ratio (OR) and 95 percent confidence intervals (CIs) under Hardy-Weinberg disequi-librium using various genetic models. The χ2 test was also utilized to compare the genotype and allele dis-tribution of these candidate gene polymorphisms between the case and control groups. The results of our study are particularly intriguing as they indicate that individuals with an AA genotype for the leptin gene are significantly more likely to have four additional functional teats (OR=7.7143, 95 percent CI=1.6025, P-value=0.0108). Additionally, the results in a dominant model (AA vs. GA+GG) revealed an OR of 0.1296 and 95 percent CI of 0.02669–0.6240, with a p-value of 0.0108, further highlighting the association be-tween leptin genotypes and supernumerary teats (SNT). Moreover, the risk of having more than four func-tional teats is heightened by the GG genotype in the FBP3 gene (P<0.13). However, our analysis did not uncover any additional significant correlation between the polymorphism of another investigated candidate gene and supernumerary teats (SNT). Overall, this topic presents an intriguing area that warrants further research to gain a comprehensive understanding of the biological mechanisms governing the development of four functional teats in ewes with large litter sizes. Manuscript profile