A FecX-mutated allele called FecX(R) in the BMP15 gene has been described in Rasa aragonesa sheep. FecX(R) causes increased prolificacy when heterozygous and sterility when homozygous in ewes. However, highly prolific ewes without the FecX(R) allele have been found in this breed. Therefore, a genome-wide association study (GWAS) was performed in 158 ewes (tail H: N = 73, mean prolificacy +/- standard deviation = 2.14 +/- 0.26; tail L: N = 85, mean prolificacy = 1.06 +/- 0.08) with the Ovine HD SNP BeadChip. In this analysis, the FecX(GR) allele was found to have genome-wide significance associated with prolificacy, first described in the Grivette sheep breed. We also identified a novel polymorphism in exon 2 of BMP15 in 9 high prolific ewes by Sanger sequencing. This new mutation, called FecX(RA), is a SNP (Oar3.1_X: g. 50970948C > T; NM_001114767.1: c.1172C > T) that produces an amino acid substitution (ENSOART00000010201: p.T400I) that is predicted to be deleterious and to alter the predicted secondary structure of the mature protein. To confirm if this SNP had any the effect on prolificacy, we genotyped sires with known EBVs (Estimated Breeding Values), finding one hemizygous sire for the FecX(RA) allele with the highest EBV in the breeding program (effect on litter size at + 0.39 lamb per lambing). A very low frequency, ranging from 0.13 to 2%, was found for the FecX(R) and FecX(RA) alleles in 3428 animals belonging to four different flocks. Finally, an association study was performed to validate and quantify the effects of the FecX(GR) and FecX(RA) alleles. Significant increased prolificacy of 0.52 +/- 0.05, 0.42 +/- 0.05 and 0.32 +/- 0.01 were found when comparing FecX(GR), FecX(RA) and FecX(R) heterozygous ewes to wild type homozygous ones. These effects are of the same order of magnitude as the effect of most of other known major genes for prolificacy. Only significant differences between FecX(GR) and FecX(R) were found among the three alleles associated with increased prolificacy. However, we cannot confirm the effect of the FecX(RA) allele at homozygous state because we did not find any homozygous ewes. These results confirm that these three alleles in the BMP15 gene that affect prolificacy co-segregate in Rasa aragonesa sheep. (C) 2020 Elsevier Inc. All rights reserved.