Nematodes were sequenced from soil samples collected across Australian cropping regions to determine what Pratylenchus species are present in each region and whether there is genetic diversity in populations that can affect the reliability of the PreDicta B assays. DNA was extracted from single nematodes and the ITS region was sequenced and used to identify the species and check for sequence variation in the diagnostic site. More than 2500 single nematodes were isolated from 455 soil samples, including 71 samples from NSW, 81 from Qld, 64 from SA, 47 from Vic and 182 from WA. Results from 1273 sequences show that P. neglectus (669), P thornei (230) and P. quasitereoides (164) were the most prevalent species with P. neglectus and P. thornei widespread across the cropping regions. P. quasitereoides and P. penetrans were almost exclusively found in WA which is consistent with previous observations. All DNA identified as from P. neglectus, P. thornei, P. quasitereoides and P. penetrans by sequencing were accurately detected by the corresponding PreDicta B assay except in 6 instances (3 P. neglectus and 3 P. thornei). In addition, none of the DNA from other Pratylenchus species was detected by any of the PreDicta B assays. The results show that the genetic diversity in the portion of the ITS sequence used as diagnostic target is low validating its choice as a reliable diagnostic target for Pratylenchus detection. Furthermore, no variant or species other than the main ones was found to be widespread and susceptible to represent a threat to the Australian grains industry indicating the current PreDicta B assays are presently sufficient to assess Pratylenchus risk.