The Sandhills of Nebraska is a complex grassland ecosystem, covering 50,000 km² in central and western Nebraska, predominantly virgin grassland. To date, little information is available on the diversity of Rhizoctonia spp. from the Sandhills. The objective of this study was to evaluate the diversity of Rhizoctonia spp. on major grasses in the Sandhills and determine pathogenicity of isolated strains, including their potential to cause disease in row crops. In 2016 and 2017, four dominant native grass species, namely, sand bluestem, little bluestem, prairie sandreed, and needle-and-thread, and soil samples were collected from 11 virgin soil locations in seven counties in the Sandhills. Fungi were isolated from discolored roots and baited from soil samples. A total of 17 isolates were collected: 5 isolates of Rhizoctonia solani AG-F; 2 isolates each from R. solani AG-B, AG-C, AG-3, and AG-4; 1 isolate of AG-L; 1 isolate of Rhizoctonia zeae. Pathogenicity was assessed using a rolled towel assay on soybean variety ‘CR2603’ and in the greenhouse on soybean, sand bluestem (Andropogon hallii), and needle-and-thread (Hesperostipa comata) grasses. Isolates of binucleate Rhizoctonia spp. were pathogenic on all grasses but did not cause significant necrosis on soybean. Isolates of AG-C and AG-3 caused severe necrosis on both grasses and soybean. These results suggest grasses in the Sandhills harbor unique AGs, some with cross-pathogenicity to cultivated crops.