Onion (Allium cepae L.) is a biennial important vegetable crop worldwide. Root and basal rot of onion caused by Fusarium oxysporum f. sp. cepae is a soilborne disease causing tremendous losses in onion in the field and also during storage at Iran. In the present study, 26 monoclonal isolates of F. oxysporum f. sp. cepae were obtained from infected onion plants showing root and basal rot from the main onion growing regions of Iran, including, Azerbaijan, Isfahan and Fars provinces. In this study, random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and virulence studies were conducted to analyze 26 F. o. f. sp. cepae isolates. Ten RAPD primers, including OPA-03, OPB-17, OPC-08, OPC-9, OPP-16, OPP-17, OPP-18, were used for genetic analysis polymorphisms among the 26 FOC isolates. For the ISSR analysis, out of nine primers from the initial screening ISSR primers, only five primers were chosen and characterized. RAPD analysis was performed in three replications to confirm the consistency of amplification and only repeatable bands were scored. Monomorphic and polymorphic bands were considered as binary characters and were scored as 1 for presence and 0 for absence of DNA bands. The scores were then entered into a matrix for analysis by numerical taxonomy and multivariate analysis system, NTSYS-pc 1.8 program. The similarity matrix was calculated using Jaccard’s similarity coefficient. Clustering was performed using the unweighted pair group method using arithmetic averages (UPGMA) to generate the dendrogram. Application of ten RAPD primers generated a total of 86 consistently amplified fragments (200-3000 bp), of which 83 (96.51 %) fragments were polymorphic. Among the primers, OPP-17 and OPC-9 offered the highest and lowest number of amplified fragments, respectively. The similarity among 26 F. o. f. sp. cepae isolates ranged from 69 to 100 % with the mean value of the Jaccard’s similarity coefficient 0.84. The five ISSR primers generated a total of 24 consistently amplified DNA bands, in which 87.5 % were polymorphic. The average number of bands per primer was 4.8 which ranged in size from approximately 200 to 800 bp. The similarity index was ranged from 32 to 100 % with the mean value of the Jaccard’s similarity coefficient 0.71. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion, however virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.