This study aimed to identify genomic regions associated with prolificacy in Santa Inês sheep raised in tropical conditions. The prolificacy of the dam was defined as single (only one lamb born per ewe per lambing) or multiple (more than one animal born per ewe per lambing). After quality control of phenotypic data, 1584 lambing records of 715 females occurred between the years 2000 and 2018 were used. The animals were genotyped with the OvineSNP50 BeadChip panel (Illumina Inc.). After quality control of genomic data, information of 46,714 SNPs and 388 samples and females was used for the subsequent analyses. The single-step GWAS (ssGWAS) methodology was used to estimate the effects of genetic markers and their association with the prolificacy. A total of 21 windows of 10 adjacent SNPs that explained at least 0.5% of the additive genetic variance for prolificacy were identified. In such regions, genes associated with different reproductive functions in the female were found: CACNA1E, NTRK1, PLCH1, SMAD3, CENPF, TOPBP1, IL33, DRD2, MID1, HCCS, and ARHGAP6. Some candidate regions related to prolificacy harbor genes that were not previously described and genes without known functions. These results can help to identify genes associated with prolificacy and could be used in genomic reproductive studies on prolificacy, as well as in the selection of the most prolific ewes in the population.