Lentil((Lens culinaris M.), as one of the most important members of the fabaceae plays fundamental role in the nutrition of the people in developing countries due to its high protein contant.Therefore, diversity is very important for the management of breeding programs. Hence evaluation of genetic diversity for exploitation of variation is essential for breeding projects and also helps breeding to select favorite parents in hybridizations. In this study, the genetic variation of 23 genotypes was assessed using ISJ markers. Based on molecular data,165 bands were detected and 117 bands were polymorph. The mean number of bands was 9.1 bands per primer using ISJ, the fragment size varied withen a significantaly narrower range(150-2500bp). The similarity matrix was subjected to cluster analysis by the unweighted pair-group method(UPGMA). The PIC value was ranged 0.201 to 0.433 and the MI index averaged(1.93). In the ISJ marker the UPGMA cluster diagram have showed 4 major clusters. According to similarity matrix, the least similarity belonged to gachsaran and F2006-6L and the highest similarity belonged to F2004-53L and F200456L. The results showed that intron-exon splice junction (ISJ) markers is an effective method for analysis of genetic variation among lentil germplasm.