Expressed sequence tag-simple sequence repeat (EST-SSR) markers were used to analyse genetic diversity among three Lens species. The SSR loci amplified successfully in wild species, with 94·82% transferability in Lens culinaris subsp. orientalis, 95·4% in Lens nigricans, 98·81% in L. culinaris subsp. odemensis, 94·82% in L. culinaris subsp. tomentosus and 96·55% in Lens ervoides. Ninety-nine alleles (average 3·41 alleles/locus) were detected by 29 SSR markers. Based on the unweighted pair group method with arithmetic mean cluster analysis, all the genotypes were grouped into three clusters at a similarity level of 0·30. The diversity analysis indicated no species-specific clustering of the wild and cultivated species. Wild species L. nigricans and L. culinaris subsp. odemensis, L. culinaris subsp. orientalis and L. ervoides were grouped in Cluster I, whereas the Mediterranean land races of L. culinaris subsp. culinaris and L. culinaris subsp. tomentosus formed a separate group in Cluster II A. Cluster II B comprised L. ervoides, L. culinaris subsp. orientalis and L. culinaris subsp. culinaris. Clusters II C, II D and II F included cultivated Indian lentil genotypes. Cluster II E comprised Indian and Mediterranean germplasm lines. Cluster II F included three early maturing germplasm lines, whereas Cluster III included only two germplasm lines. The functional annotation of SSR-containing unigenes revealed that a majority of genes were involved in an important transport-related function or were a component of metabolic pathways. A high level of polymorphism of EST-SSRs and their transferability to related wild species indicated that these markers could be used for molecular screening, map construction, comparative genomic studies and marker-assisted selection.