The nematode Deladenus siricidicola is the primary biological control agent of Sirex noctilio, a globally invading woodwasp pest of Pinus species. Preliminary studies on the diversity of populations of D. siricidicola revealed very low diversity in the Southern Hemisphere where they have been introduced for the purpose of biological control. The potential to augment biocontrol efficacy by increasing genetic diversity in biocontrol programs motivated this study, which investigated the patterns of genetic diversity in D. siricidicola across eight countries, including the presumed native range (Spain), areas of accidental introduction (Canada and the USA) and countries D. siricidicola has been intentionally released (Argentina, Brazil, Chile, New Zealand and South Africa). Nematodes were screened using mitochondrial COI sequence data and twelve microsatellite markers. Analyses of these data identified three distinct lineages from North America (Lineage A), the Southern Hemisphere (Lineage B) and Spain (Lineage C). Strains from Chile were an exception as they appear to represent an admixture of lineages A and B. This suggests a common origin of populations throughout the Southern Hemisphere, with a second introduction from North America into Chile. The introduction into North America is distinct from that in the Southern Hemisphere and probably originated from Europe. It is evident that substantial genetic diversity exists in D. siricidicola globally, which could be exploited to augment the reduced diversity in some populations used in biocontrol programs.