Rhizoctonia solani, a common pathogenic fungus of potato plants, causes stem cankers and black scurf. This study aims to investigate the genetic diversity of Rhizoctonia solani isolates; causes stem cankers and black scurf diseases from various potato fields. This study utilized sequence analysis and restriction digestion of PCR-amplified internal transcribed spacers (ITS1, ITS2) and the 5.8S region of ribosomal DNA (rDNA) genes. The amplification of the ITS1-5.8S-ITS2 region using universal primers (ITS1 and ITS4) yielded an approximately 700 bp amplicon, revealing notable sequence heterogeneity among the isolates examined. Subsequent digestion of the amplified products with HaeIII, HinfI, and XbaI restriction enzymes unveiled distinct banding patterns across the tested isolates, distinguished by varying numbers of bands and fragment sizes. This study underscores the efficacy of DNA sequencing and ITS restriction fragment length polymorphism (RFLP) as valuable molecular techniques for exploring nucleotide polymorphisms within genetically diverse R. solani isolates.