Expressed sequence tags simple sequence repeats (EST-SSRs) are important sources for investigation of genetic diversity and molecular marker development. Similar to genomic SSRs, the EST-SSRs are useful markers for many applications in genetics and plant breeding such as genetic diversity analysis, molecular mapping and cross-transferability across related species and genera. In spite of low polymorphism, these markers show variation in the expressed part of the genome. In this study, Medicago truncatula EST-SSRs were used for investigation of transferability between M. truncatula and some chickpea (Cicer arietinum L.) genotypes, also genetic diversity between used chickpea genotypes was determined. In this research, 650 M. truncatula ESTs were searched to find simple sequence repeats (SSRs). A total of 131 EST-SSRs were contained di-and trinucleotide motif SSRs. In this study, thirty pairs of primers were designed to amplify over 10 chickpea genotypes. Thirteen primer pairs (43%) generated reproducible bands in at least one chickpea genotype that eight bands (61.5%) were polymorphic in the chickpea genotypes. A total of 24 alleles were amplified with an average of 3 alleles per primer. The average of polymorphism information content (PIC) was 78.75% and transferability across M. truncatula and C. arietinum was 43.32%. The results indicate that the developed EST-SSR markers from M. truncatula as a model plant are valuable genetic markers for legume species such as chickpea. In addition to suitability of EST-SSR markers for genetic diversity analysis, their broad range of transferability also proved their potential for comparative genomics studies.