Cereal cyst nematode (CCN) is a root disease caused by the pathogen Heterodera avenae Woll. that significantly reduces wheat (Triticum aestivum L.) grain yields in temperate countries. The Cre3 gene, located on chromosome 2DL, provides high levels of resistance to the Australian pathotype and isolates from Syria and Algeria, and has become available to wheat breeders. Selection for lines carrying the Cre3 gene in Australian wheat breeding programs is currently based on a dominant PCR marker (Cre3spf/r) diagnostic for the Cre3 gene. However, this marker has limitations that increase the cost and reduce selection efficiency in screening early-generation breeding lines. Such limitations would be minimised by the identification of a microsatellite marker linked to the Cre3 gene. We have constructed 2 genetic linkage maps of wheat chromosome 2DL and identified microsatellite markers mapping closely to the diagnostic Cre3spf/r marker. These closely linked markers were validated in a diverse range of germplasm, and one microsatellite marker, Xgwm301, which mapped 4 cM from Cre3spf/r, was shown to be highly associated with the presence of the Cre3 gene. Amplification conditions for the Xgwm301 locus were optimised, and its use in marker-assisted selection to identify Cre3 CCN-resistant wheat in the Australian Grain Technologies breeding program is demonstrated.