Rhizoctonia is a widespread soilborne fungus in different agroecosystems worldwide. It has been reported as a destructive fungal pathogen that caused various types of diseases on a wide variety of crops. The aim of this study was to characterize Rhizoctonia spp. isolated from various crops. We obtained 37 Rhizoctonia isolates from rice, corn, spinach, chrysanthemum, and chili grown in various locations in Peninsular Malaysia. A total of 35 isolates were classified under Rhizoctonia solani (multinucleate) and two isolates belonged to binucleate Rhizoctonia (BNR) according to the number of nuclei and morphological characteristics. Based on rDNA-ITS sequences and PCR profiling using AG group-specific primers, these isolates were identified as R. solani AG-1 IA (75.6%), AG-1 ID (10.8%), AG-2-2 IV (2.7%), AG-4 HG-I (5.4%), BNR AG-Fa (2.7%), and AG-A (2.7%). A specific primer of binucleate Rhizoctonia AG-Fa was designed and its specificity was evaluated by PCR. Further investigation using tef-1α gene confirmed the AG group identities for these isolates. Clustering analysis of molecular markers (OPA13 and OPE-6, 2080 iPBS, and 2249 iPBS and ISSR 3) supported the AG concept rather than geographical origin or plant host. Pathogenicity tests on the leaves of rice and corn showed that R. solani AG-1 IA rice isolates cause necrotic lesions greater than AG-1 IA corn isolates. Rhizoctonia solani AG-1 IA, AG-2-2IV, and AG-4 HG-I isolates cause necrotic lesions greater than BNR isolates on seedling of cabbage, cauliflower, and radish. To the best of our knowledge, this is the first study on morphology and molecular characterization of Rhizoctonia spp. isolated from various crops and regions in Malaysia.