Technical Abstract: Our objectives were to develop molecular markers capable of characterizing the diversity of the Pyrus collection of the U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository. Simple sequence repeat (SSR) or microsatellite markers have become the marker of choice for genotype identification. A method was developed to identify microsatellite-containing SSRs from existing pear GenBank sequences. We screened 366 genomic, mRNA and expressed sequence tag (EST) Pyrus nuclear sequences and identified 47 that contained SSRs. Primer pairs were designed for 18 sequences and the optimum annealing temperature was determined by gradient PCR. The SSR primers were able to amplify a product in eight cultivars of P. communis, three cultivars of P. pyrifolia and one Pyrus hybrid ('Nijisseiki' x 'Anjou'). Three primer pairs amplified fragments larger than the expected size and were not pursued further. The remaining 15 primer pairs amplified fragments of the expected size, and 10 of them appeared to show polymorphism. These 10 polymorphic loci are being used to characterize the Pyrus collection at the NCGR.