The molecular characterization of Pratylenchus species determined from D2-D3 of 28S rDNA, ITS of rDNA, and COI of mtDNA regions revealed four Pratylenchus species from North Dakota, P. scribneri, P. neglectus, Pratylenchus sp. (ND-2016 isolate HG51), and Pratylenchus sp. (ND-2017). They were clustered in four separate clades in the phylogenetic trees indicating the divergence among species. P. scribneri and Pratylenchus sp. (ND-2016 isolate HG51) were closely associated and Pratylenchus sp. (DH-2017) was closely related to Pratylenchus sp. (ND-2016 isolate HG51). However, P. neglectus was not closely associated with the other three species. Moreover, resistance evaluation of ten corn hybrids to Pratylenchus scribneri, P. neglectus, and Pratylenchus sp. (ND-2017) revealed that 1392 VT2P was moderately resistant to three Pratylenchus species. PFS74K89 and 4913 VT2RIB were moderately resistant to two of the three Pratylenchus species. X5B-8801, DK 43-46, and DKC 44-13 were susceptible to two of the three Pratylenchus species.