The woodwasp Sirex noctilio Fabricius, along with its obligate symbiotic fungus Amylostereum areolatum (Chaillet ex Fr.) Boidin, is amongst the most damaging invasive species to many commercial pine plantations. The most effective biocontrol agent for management of this woodwasp has been the nematode Deladenus siricidicola Bedding. Before this agent can be used in North America, answering key questions about its interaction with native siricids and other strains of the nematode is essential, as would be the need to track its spread after release. The aim of this study was to develop tools to differentiate between the North American D. siricidicola isolates and the Southern Hemisphere Kamona strain of this species. We sequenced a region from ribosomal DNA and the cytochrome oxidase subunit 1 and developed a PCR–RFLP method based on a single nucleotide polymorphism flanking a microsatellite sequence. These markers will be useful for science-based operational biocontrol of S. noctilio.