Rhizoctonia solani anastomosis group (AG)-2-2IIIB and AG-4HGI are the main pathogens causing sugar beet seedling damping-off and crown and root rot disease. In this study, 1232 loci of simple sequence repeats (SSRs) were obtained via transcriptome sequencing, with 592 from AG-2-2IIIB and 640 from AG-4HGI. Fourteen and twenty loci of SSRs were selected for studying the genetic structure of the AG-2-2IIIB and AG-4HGI populations, respectively. A population of 134 strains of AG-2-2IIIB and 145 strains of AG-4HGI, sampled from three geographic regions in China, indicated that both AG-2-2IIIB and AG-4HGI had a high level of genetic diversity, and that the selected SSR markers could reliably capture the genetic variation. Genetic analysis indicated that the individual strains of AG-2-2IIIB and AG-4HGI randomly mated within their respective population, and that a considerable degree of inbreeding was present among the populations. High to moderate gene flow and low to moderate population subdivision were detected among the populations of AG-2-2IIIB and AG-4HGI, which indicated that weak differentiation existed in these two subgroups. In addition, a founder effect (genetic drift) or a bottleneck effect was inferred to have occurred in the AG-4HGI population. This study provides the first analysis of the population genetic structure of AG-2-2IIIB and AG-4HGI associated with sugar beet seedling damping-off and crown and root rot disease, and the present results offer useful guidance for developing effective integrated disease management.