Abstract: In order to investigate species and distribution of the Potato Cyst Nematode (PCN), Globodera spp., present in Tunisia, soil samples were collected from 118 different potato fields in 6 different regions. Identification of Globodera species was based on some morphological criteria and confirmed by PCR-RFLP. The RAPD was used to compare Tunisian and Europeans PCN populations to reveal a possible phylogeny between them and then determine the origin of infestations. This is the first report of molecular characterisation and polymorphism of PCN populations in Tunisia. Biological tests using resistant and susceptible cultivars of potato were done in vitro on G. rostochiensis populations to determine the pathotypes. Analysis showed that 37.28% of the investigated fields were infested by PCN. Morphological and molecular analysis, showed the presence of G. pallida and G. rostochiensis having different proportions according to regions. The RAPD revealed that Tunisian G. pallida populations correspond to Pa2/3 pathotypes group and biological results proved that the G. rostochiensis populations belong to the Ro1/4 pathotype. Results from the bootstrap analysis showed a great similarity with G. pallida and G. rostochiensis groups of European populations, evidence that Tunisian populations of PCN are derived from importation and exchange of potato seeds.