Globodera tabacum is a little-known species complex of specialized plant-parasitic nematodes. In this study we investigated for the first time the sequence variability of five effectors: three genes coding for cell wall degrading enzymes (Pel1, Pel2 and ExpB3) and two genes coding for CLE peptides (CLE1 and CLE4) among a set of populations representing the different subspecies described. Substantial variability was observed in the sequence dataset which is consistent with the differential pathogenicity of the G. tabacum subspecies on a range of Nicotiana species. Using these sequence data, we were able (1) to show the presence of a novel CLE-like class in cyst nematodes, (2) to support the existence of most probably three and not four subspecies in this species complex, (3) to reveal that the subspecies G. tabacum tabacum presents the highest diversity and (4) to develop a PCR tool that allows a quick and reliable identification of this subspecies among the species complex.