To date, very limited molecular knowledge and molecular tools are available on grass pea (Lathyrus sativus L.). Herein, we present a genetic diversity analysis of 20 grass pea accessions collected from various regions of Ethiopia by means of seven newly developed Lathyrus expressed sequence tag-derived simple sequence repeat (EST-SSR) markers and four cross-transferable EST-SSRs derived from Medicago truncatula L. Forty-five alleles were detected among all the analyzed accessions. The number of alleles/locus ranged from two to seven with an average of four alleles/locus. The observed heterozygosity (Ho) ranged from 0.320 to 0.504, while the expected heterozygosity (He) ranged from 0.354 to 0.470. FST values estimated by analysis of molecular variance were 0.01, 0.15 and 0.84 for among regions, among accessions and within accessions, respectively, indicating that most of the variation (84%) resides within accession. The model-based cluster analysis grouped the accessions into three clusters grouping accessions, irrespective of their collection regions.