Rhizoctonia solani anastomosis group 4 (AG-4) is a serious pathogen causing damping off and root rot in many important crop plants. A total of 190 isolates of R. solani AG-4 HG-I were collected from host fields in five provinces of Iran. The genetic structure of this pathogen was evaluated using seven microsatellite loci, focusing particularly on geographic differentiation. Most of the multilocus genotypes (MLGTs) were unique, with few MLGTs shared among populations. High to moderate levels of gene flow among populations was indicated by low to moderate differentiation between pairs of populations based on the fixation index (FST). Gametic equilibrium of most pairs of microsatellite loci and moderate genotypic diversity were found for two out of five populations, indicating that these populations were sexually recombining in structure. High genotypic diversity, moderate clonal fractions and site-specific genotypes were consistent with mixed reproductive systems for the remaining populations. The findings of departures from Hardy–Weinberg (HW) equilibrium, gametic disequilibrium and a significant excess of homozygotes in half or more than half of the loci were probably caused by the presence of null alleles and the Wahlund effect. This is the first study to consider the population genetics of the root and crown rot pathogen R. solani AG-4.