The Pelibuey sheep has adaptability to climatic variations, resistance to parasites, and good maternal ability, whereas some ewes present multiple births, which increases the litter size in farm sheep. The litter size in some wool sheep breeds is associated with the presence of mutations, mainly in the family of the transforming growth factor β (TGF-β) genes. To explore genetic mechanisms underlying the variation in litter size, we conducted a genome-wide association study in two groups of Pelibuey sheep (multiparous sheep with two lambs per birth vs. uniparous sheep with a single lamb at birth) using the OvineSNP50 BeadChip. We identified a total of 57 putative SNPs markers (p < 3.0 × 10-3, Bonferroni correction). The candidate genes that may be associated with litter size in Pelibuey sheep are CLSTN2, MTMR2, DLG1, CGA, ABCG5, TRPM6, and HTR1E. Genomic regions were also identified that contain three quantitative trait loci (QTLs) for aseasonal reproduction (ASREP), milk yield (MY), and body weight (BW). These results allowed us to identify SNPs associated with genes that could be involved in the reproductive process related to prolificacy.