This paper reports the development of new microsatellite markers for lentil (Lens culinaris subsp. culinaris) and their use for genetic diversity analysis of a lentil core collection developed at ICARDA (Aleppo-Syria). Fourteen new markers were developed from microsatellite flanking sequences of a genomic library from a cultivated lentil accession ILL5588. The core collection used comprises 109 accessions from 15 countries representing 57 cultigens (including 18 breeding lines) from 8 countries and 52 wild types of germplasm (L. culinaris subsp. orientalis, L. culinaris subsp. tomentosus and L. culinaris subsp. odemensis) from 11 countries. Total number of alleles detected across all microsatellite loci was 182, with a mean of 13 alleles per locus. The wild accessions were rich in alleles (151 alleles) compared to cultigens (114 alleles). The genetic diversity index for the microsatellite loci in the wild accessions ranged from 0.16 (for locus SSR28 in L. culinaris subsp. odemensis) to 0.93 (for locus SSR66 in L. culinaris subsp. orientalis) with a mean of 0.66, while in the cultigens genetic diversity varied between 0.03 (locus SSR28) and 0.87 (locus SSR207) with a mean of 0.65. The cluster analysis indicated two major clusters, mainly one with the cultigens and the other with the wild accessions.