High-density SNP panels (e.g., 50,000 and 600,000 SNPs) have been used in exploratory population genetic studies with commercial and minor sheep breeds. Routine genetic diversity evaluations of large numbers of samples and panels are in general cost-prohibitive for gene banks. Lower cost panels based mostly on SNPs known to be associated with production traits of interest and may be more efficient for genetic diversity assessment and improvement of gene bank collections. The first phase of the study was to develop and validate a small SNP panel (29 SNPs) based on known prolificacy genes. SNP selection was based on known polymorphisms in major genes affecting sheep prolificacy (GDF9, BMP15 and BMPR1B), as well as new polymorphisms mined from whole genome resequencing data from GDF9 and BMP15 by the International Sheep Genome Consortium. A total of 125 animals from 15 breeds with litter size information collected by the National Animal Germplasm Program were tested. Genotyping was performed with PCR-based KASP chemistry and 27 SNPs had a call rate higher than 98%. All BMP15 and BMPR1B SNPs were monomorphic. In GDF9 8 SNPs were polymorphic - 3 are located in introns and 5 in exons. Three SNPs were non-synonymous AA changes. No significant allele frequency differences were found for GDF9_G4:E241K among from single or multiple births. The GDF9_G6:V332I allele was related to high prolificacy in 5 breeds with the following frequencies: Lincoln (0.416), Polypay (0.32), Navajo Churro (0.27), St Croix (0.125) and Suffolk (0.083). Allele GDF9_G7:V371M was found only in Polypay at a frequency of 0.35 which agrees with recent studies with the hyper-prolific Norwegian White Sheep. The other 10 breeds did not differ significantly. Our results suggest that the developed panel will be useful to identify possible genes/mutations associated with prolificacy in worldwide sheep breeds. Additional SNPs will be included as more information becomes available. In the next phase of this study, SNPs related to other economically important traits will be added to the panel to improve the characterization and management of gene bank sheep collections.