Tobacco cyst nematode is a sexually diploid species composed of three subspecies, Globodera tabacum subsp. tabacum (Gtt), G. tabacum subsp. virginiae (Gtv) and G. tabacum subsp. solanacearum (Gts). In Europe, only Gtt have been reported to date. Here, we report from a plant in a French tobacco field, the identification of a mixture between cysts of the subspecies Gtt and cysts genetically different. The objective of the present study was to determine the subspecies status of those particular cysts. To this aim, we performed CAPS analyses on two genes and a principal component analysis on microsatellite data. The results obtained with both CAPS and microsatellite markers demonstrate for the first time the occurrence of G. tabacum subsp. virginiae in France.