Stem canker and black scurf of potato (Solanum tuberosum L.) caused by Rhizoctonia solani Kühn are important and epidemic diseases in potato-growing regions worldwide, including Iran. In this study, 120 isolates were retrieved from infected stem canker from six potato- growing regions in Iran (Isfahan, Ardebil, Fars, Hamedan, Kurdestan and Kerman). Out of these, 30 isolates were selected as representatives for genetic and virulence analysis. The isolates were analyzed by one sequence analyzes of the ITS-rDNA region, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), as well as virulence studies. Based on sequence analysis of the ITS-rDNA region, all 30 isolates were assigned to the anastomosis group (AG) and all were assigned to AG-3 PT. Cluster analysis using the unweighted pair group method with the arithmetic averages (UPGMA) method for both RAPD and ISSR markers revealed that they were divided into three main groups, with no correlation to geographical regions of the isolates. Pathogenicity tests showed that all isolates were pathogenic on potato cv. Agria; however, virulence variability was observed among the isolates. The grouping based on RAPD analysis and virulence variability was not correlated.