Plant parasitic nematodes are a major biotic cause of wheat-yield loss in temperate wheat-growing regions. A major strategy to develop resistance to root-lesion nematodes (RLNs) in wheat is to assess and then exploit their natural genetic variation. This study examines RLN (Pratylenchus thornei) resistance in I Middle Eastern landrace (AUS4930 7.2) and 1 synthetic hexaploid wheat, CROC_1/AE.SQUARROSA (224)//OPATA (CROC), using F-2 and F-9 populations generated by crossing AUS4930 7.2 and CROC with the susceptible cultivar Pastor, and inoculating these crosses with P. thornei in greenhouse trials. Wheat microsatellite markers linked to previously identified quantitative trait loci (QTLs) for resistance to P. thornei and P. neglectus were used to screen both populations. In the AUS4930 7.2 x Pastor population. resistance loci on chromosomes 1B, 2B, and 6D were detected. Similarly, in the CROC x Pastor population, 2 resistance loci, located on chromosomes 1B and 313, were identified. Interestingly, a resistance locus located on chromosome 6D was not detected. More detailed mapping is required in these 2 populations, developed using new RLN resistance sources, to determine whether the QTLs identified on these chromosomes are the same, are allelic, or are linked to different resistance loci from those previously identified, and to determine whether these 2 sources contain other novel resistance loci.