Fertility traits have the greatest financial impact on sheep production. In this study we aimed to characterize polymorphisms of the KISS1 receptor gene (KISS1R), also known as the G-protein-coupled receptor 54 gene (GPR54) that is reported to be involved in the control of puberty and reproductive function. Genomic DNA were obtained from 156 ewes of pure Mehraban and Shal Iranian native sheep and their crossbreeds with Romanov. The exploration of polymorphisms of the KISS1R/GPR54 gene (GenBank No: HM135393.1) was performed by single-strand conformation polymorphism analysis (SSCP) and Sanger sequencing. Seven single-nucleotide polymorphisms (SNPs) including g.396 T > G, g.456 T > C, g.475C > A, g.571 A > C, g.3431C > A, g.4108 G > A and g.4123C > A, were observed in the four breeds. Among these SNPs the g.3431C > A in the exon 4 was the only amino acid altering variant (p.195 Phe > Leu). Subsequent statistical analysis revealed that the minor A allele at this position had a significant (P < 0.01) negative effect on litter size (LS) and birth weight (BW) and could be considered as a causal mutation impairing these traits. No significant (P > 0.05) allelic association with the studied traits was found at the position g.396 T > G, g.456 T > C, g.475C > A and g.571 A > C. In contrast, carrier ewes of the SSCP pattern F (homozygous reference; g.4108 G/G, g.4123 C/C) showed a significantly (P < 0.01) higher LS than ewes carrying the patterns G (heterozygous; g.4108 G/A, 4123 C/A) or E (homozygous variant; g.4108 A/A, g.4123 C/C). The results of the present study provide additional evidences on the potential role of the KISS1R/GPR54 gene in controlling reproductive traits and particularly prolificacy in sheep.