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Background: Litter size plays a crucial role in determining profitability in the sheep industry. Breeding sheep with high litter sizes could be enhanced by selecting candidate genes. One gene affecting sheep's reproductive performance is the hormone-sensitive lipase (HSL) gene. As a result, this study investigated whether the HSL gene variation influenced the fertility of Awassi ewes. The genomic DNA was extracted from 52 singleton ewes and 48 twin ewes. The HSL gene exon 9 (278 bp) was amplified using polymerase chain reaction (PCR). Results: Study results revealed two genotypes identified in the 278-bp amplicons: GG and GA. Molecular sequence analysis identified a novel mutation in the GA genotype 216G > A. The statistical analysis revealed a significant association between the single nucleotide polymorphism (SNP) 216A > G and reproductive performance. Ewes with the SNP 216G > A genotype exhibited significantly increased litter sizes, twinning rates, lambing rates, and fewer days to lambing compared to ewes with GG genotypes (P ≤ 0.05). The logistic regression analysis results provided strong evidence that the 216G > A mutation significantly increased litter sizes. Conclusions: This study concluded that variant 216G > A SNP positively impacts Awassi sheep reproduction. There is a higher litter size and more prolificacy in ewes with the 216G > A SNP than in those without the SNP. [ABSTRACT FROM AUTHOR]