Potato leaf blight disease caused by Ulocladium atrum (Syn. Stemphylium atrum) is an important and epidemic disease in potato-growing regions of Iran. In this study, 30 isolates of the disease were collected from the main potato-growing regions of Iran and were analyzed on the basis of morphological characterization and pathogenicity. Based on morphological characteristics, all isolates were identified as U. atrum. Pathogenicity studies indicated that all 30 isolates were pathogenic on potato “Agria” to varying degrees. Five U. atrum isolates causing potato leaf blight disease, obtained from the Plant Pathology Laboratory, Isfahan Research Center for Agriculture and Natural Resources, Isfahan, Iran, were also examined in this study. A total of 35 isolates were genetically analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Cluster analysis using the un-weighted pair group method with the arithmetic average (UPGMA) method for RAPD marker revealed no clear grouping of the isolates obtained from different geographical regions. The groupings, based on morphological characteristics, virulence variability and RAPD analysis, were not correlated. Cluster analysis using Jaccard’s coefficient for ISSR divided the U. atrum isolates into four main groups, in which there was no significant correlation between the isolate groupings regarding their geographic location and pathogenicity. Using molecular techniques genetic variability was detected among the accessions, with cophenetic correlation coefficients (CCC) of 0.80 for RAPDs and 0.89 for ISSRs. The RAPD and ISSR marker results corresponded well, with a correlation of 0.55.