The root lesion nematode, Pratylenchus neglectus, is an economically damaging pathogen of wheat and other crops. The development of P. neglectus-resistant wheat cultivars would be greatly accelerated through the use of molecular markers, as resistance phenotyping is extremely time-consuming. A greenhouse bioassay was developed to identify resistance phenotypes of doubled-haploid populations. Bulked-segregant analysis was used to identify AFLP markers linked to P. neglectus resistance in the wheat cultivar Excalibur. One resistance-linked AFLP marker was mapped close to chromosome 7A RFLP markers in a densely-mapped Cranbrook/Halberd population. One of the chromosome 7A RFLP probes, cdo347, was genotyped in the Tammin/Excalibur population segregating for response to root lesion nematode and showed 8% recombination with the P. neglectus resistance gene Rlnn1. The marker Xcdo347-7A was validated on Excalibur-and Krichauff-derived DH populations segregating for Rlnn1 and showed 14% and 10% recombination, respectively, with Rlnn1 in these populations.