Rhizoctonia spp. are the main causal agents of root and crown rot on sugar beet. In this study, isolates of Rhizoctonia spp. were obtained from diseased sugar beet in Iran over 2 years. Of 68 isolates, 61 were R. solani and 7 were R. cerealis. The anastomosis group (AG) of all isolates was determined on glass slides against the testers. Characterization of intraspecific groups (ISGs) of R. solani isolates revealed that, of 61 isolates, 43 were AG2-2 IIIB and 18 were AG2-2 IV. Amplified fragment length polymorphism (AFLP) analyses were used to investigate genetic structure of Rhizoctonia populations. Principal coordinate plots and cluster analysis differentiated R. solani from R. cerealis isolates and separated the R. solani isolates belonging to different ISGs. AFLP data indicated that the R. solani and R. cerealis populations are not clonal. Analysis of molecular variance in AG2-2 IIIB isolates showed that geographic region was the main factor determining genetic structure of the populations. Sampling year had no significant effect on the genotypes. Pathogenicity tests on Beta vulgaris ‘FD0432’ revealed that R. solani AG2-2 IIIB and AG2-2 IV isolates were more virulent than R. cerealis.