In 2014, 2000 plants showing root rot symptoms were collected from 200 commercial soybean (Glycine max L.) fields in South Dakota, USA. One thousand one hundred thirty fungal isolates obtained from the samples were identified as Fusarium species by morphology and sequencing of the translation elongation factor 1-alpha gene. Fifty-one percent were F. graminearum, 30.0% were F. acuminatum, 8.0% belonged to the F. oxysporum species complex, 3.4% were F. armeniacum, 2.0% were F. proliferatum, 1.6% were F. virguliforme, 1.2% belonged to the F. solani species complex, 0.4% were F. nanum (syn. F. incarnatum-equiseti species complex), 0.1% were F. bulbicola (syn. F. fujikuroi species complex) and 0.1% were F. commune. The pathogenicity of 57 isolates was evaluated on the soybean ‘Asgrow 1835’ under greenhouse conditions. Root rot severity was rated 14 days after inoculation on a 1-to-5 scale and expressed as relative treatment effects (RTE). Nine isolates of F. oxysporum, and one each of F. armeniacum and F. commune, caused significantly greater RTE than in the non-inoculated control. Twenty-one accessions were screened for resistance in the greenhouse using one isolate each of F. graminearum, F. proliferatum and F. sporotrichioides. Accession PI361090 was less susceptible to F. graminearum and F. proliferatum than the susceptible controls, ‘Williams 82’ and ‘Asgrow 1835’. All accessions were more susceptible to F. sporotrichioides. These findings suggest that 11 Fusarium species or species complexes are pathogenic to soybean in South Dakota, and PI361090 may be a useful source of resistance to F. graminearum and F. proliferatum.