This study was conducted to assess the genetic variation of forty chickpea (Cicer arietinum L.) genotypes including lines, native accessions and cultivars based on ISSR markers and resistance to Fusarium wilt. For study reaction to Fusarium wilt, genotypes were inoculated with three aggressive isolates of Fusarium oxysporum f.sp ciceris in three separate experiments. These experiments were conducted in complete randomized design (CRD) with three replications in sick pot culture under controlled greenhouse condition. Results showed significant differences among genotypes for disease severity after inoculation. Cluster analysis based on disease severity test classified genotypes into four distinct clusters (highly resistance, resistance, susceptible and highly susceptible). For genetic assessment, we screened and optimized nine primers based on analysis of ISSR markers among tested primers. Selected primers produced 61 clear and reproducible bands that 44 bands were polymorphic (72.1 %). Measures of marker utility, average band informative (Ibav= 0.52) and marker index (Ml= 2.53) indicated that ISSR molecular marker is effective tool for genetic studies. Based on ISSR analysis the highest genetic similarity measured between F-01-47 line and KC-2170 I I native accession and lowest genetic relationship was observed between Kaka cultivar and F -02- 21 line. Cluster analysis based on ISSR data in 0.7 level of similarity divided genotypes in to ten clusters.