Reproductive traits (especially litter size) are usually characterized by low heritability, and thus, phenotypic selection is often ineffective and slow. In order to improve fertility characteristics such as ovulation rate and litter size, it seems more effective to select breeding animals based on their genotype. The aim of the study was to use genome-wide association study (GWAS) in three sheep breeds to identify the genetic variants affecting the litter size in sheep. The study allowed us to identify one genome-wide significant SNP (rs402032081-located in ephrin type-A receptor 6, EPHA6) showing an association with litter size in Polish Mountain Sheep. We suggest that the EPHA6 gene can be a candidate gene for prolificacy trait in selected breeds of sheep; however, it needs further functional data for validation.