Abstract Heat Shock Transcription Factor 1 (HSF1) is a major regulator of cellular stress response and thermotolerance in livestock species. Genetic polymorphisms within the HSF1 gene may influence adaptability, productivity, and resistance to environmental stress in sheep. This study investigated single nucleotide polymorphisms (SNPs) of the HSF1 gene in Balami, Uda, and Yankasa sheep breeds reared in Kano State, Nigeria. A total of ninety (90) sheep comprising 30 animals from each breed were sampled. Blood samples were collected through jugular venipuncture and genomic DNA was extracted using a commercial extraction kit. Polymerase Chain Reaction (PCR) amplification of the HSF1 gene was performed followed by sequencing analysis for SNP identification. Genetic diversity indices including allele frequency, genotype frequency, observed heterozygosity, expected heterozygosity, polymorphic information content (PIC), and Hardy–Weinberg equilibrium were analyzed using standard population genetic procedures. Three SNP loci were identified within the amplified region of the HSF1 gene: g.234A>G, g.489C>T, and g.612G>A. Significant variations (p<0.05) in allele and genotype frequencies were observed among the three breeds. Yankasa sheep exhibited higher heterozygosity values (0.61) compared to Balami (0.53) and Uda (0.49). The PIC values ranged from 0.31 to 0.46, indicating moderate polymorphism within the studied populations. Phylogenetic analysis revealed closer genetic similarity between Balami and Uda breeds than with Yankasa. The identified SNPs may serve as useful molecular markers for genetic improvement and heat tolerance breeding programs in Nigerian sheep populations.