Simple Summary:The NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on litter size in sheep are unknown. In this study, we conducted population genetic and association analyses on five NLRP5 and NLRP9 loci of sheep. Our results suggested that a mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as the growth differentiation factor 9 (GDF9), whereas a mutation in g. g.59030623T > C may enhance the NLRP9-combining capacity with these proteins. Consequently, these mutations may lead to differences in ovulation rate and even litter size. Overall, this study provided useful genetic markers that can be used to improve sheep breeding.Abstract: Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY ® SNP system at the loci g.60495375A > G, g.60495363G > A, and g.60499690C > A in NLRP5, and g.59030623T > C and g.59043397A > C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy-Weinberg equilibrium (p > 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G > A in NLRP5 and g.59030623T > C in NLRP9, were highly associated with litter size. The mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T > C may enhance the combining capacity of NLRP9 with these proteins; consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals. zygotic development [1]. Additionally, a lack of NLRP5 in mouse oocytes resulted in premature activation of the mitochondrial pool, which results in mitochondrial damage that cannot be recovered by BCL2 associated X (Bax) inactivation [2], and NLRP5 knockout in mice led to female infertility [3]. Furthermore, preovulatory aging in mouse oocyte maturation decreased NLRP5 abundance, which indicated that NLRP5 has critical roles in oocyte development [4]. Notably, NLRP5, as a member of the sub-cortical maternal complex, was also found only expressed in ovine ovary and especially in the ovine oocytes in the germinal vesicle and metaphase II stage [5], which suggested their important roles in the oocyte developmental potential of sheep. However, its effects on sheep litter size are poorly understood.NLRP9 is also an important member of the NLR family. NLRP9 is highly detectable during in vitro maturation of bovine oocytes from small folli...