To elucidate the association between polymorphism of SYNE2 and litter size in sheep and provide a new locus for marker-assisted selection of high fecundity traits in sheep. A total of 384 small tail han sheep (STH) were sampled to detect single nucleotide polymorphism (SNP), and Sequenom Mass ARRAY®SNP assay was applied to genotype SNP loci of the SYNE2 gene. In this study, four SNPs were identified and that SNPs were identified that involved in amino acid changes. Population genetic analysis indicated that SYNE2 gene g.73310578G>A, g.73312791A>G showed moderate polymorphism (0.25<PIC<0.5) in Small Tail Han sheep. Furthermore, g.73310578G>A and g.73312791A>G loci were closely linked in STH (r2 > 0.33). Association analysis results showed that g.73310578G>A and g.73312791A>G SNPs significantly affected litter size (P < 0.05). In addition, the litter size of individuals with the combined genotype AA/AG was greater than that of individuals with AA/GG, GA/AG, and GG/AA genotypes in the third parity (P < 0.05). In summary, the SYNE2 gene had a positive influence on the litter size of STH sheep. The linkage of g.73310578G>A and g.73312791A>G could be used in the marker-assisted selection of the litter size of STH.