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A regional nematode survey of potato fields was conducted in the central United States during 2002 and 2003. The survey encompassed seven states and included a morphological and molecular examination of nematodes of regulatory concern from 1,929 soil samples. No regulated pest species were recovered during this survey. Meloidogyne juveniles extracted from soil were identified by mitochondrial and 18S ribosomal molecular markers. Eighty-two DNA sequences representing the two marker regions for Meloidogyne species were submitted to GenBank to facilitate evaluation of marker variability. Sufficient 18S variation was observed among some Meloidogyne species to aid in identification; however, nucleotide sequence from this highly conserved region of 18S did not discriminate among M. arenaria, M. incognita, and M. javanica. The mitochondrial gene region provided greater species discrimination and revealed intraspecific variation among many isolates. One nucleotide substitution found in a subset of M. hapla isolates from west Texas and New Mexico affected a DraI restriction site used in the PCR/RFLP diagnostic protocol. None of the mitochondrial sequence variants observed in this study compromised the PCR/RFLP identification protocol for M. chitwoodi. Additional sequence analysis is recommended for validation and evaluation of genetic markers used in diagnostic decisions.