Isoelectric focusing was used to separate proteins from cyst extracts of potato cyst nematode (PCN) populations. In a first set of assays, cyst extracts from standard populations of Globodera rostochiensis pathotypes Ro1, Ro2, Ro3, Ro2/3, Ro4, and Ro5, and G. pallida pathotypes Pa2 and Pa3, were loaded on isoelectric focusing gels. Gels were stained for superoxide dismutase (SOD), esterase, and glucose-6-phosphate isomerase (GPI). Twelve bands of SOD activity were detected, six (B1-B6) migrating towards the basic zone and the other six (A1-A6) migrating towards the acidic zone, starting from the loading point. A cluster analysis was carried out based on a data matrix that reported the presence or absence of SOD bands on the isozyme electrophoresis patterns (IEPs). Globodera spp. were clearly distinguished and, within G. rostochiensis, Ro2 and Ro4 shared a high level of similarity, respectively, with Ro3 and Ro5; moreover, Ro1 could be clearly distinguished from Ro2/3 and Ro4/5. Globodera pallida Pa2 and Pa3 also shared a high level of similarity. In contrast, esterase and GPI IEPs did not discriminate among G. rostochiensis standard pathotypes. Subsequently, 14 field populations of G. rostochiensis, five from Italy and nine from Venezuela, and three field populations of G. pallida, two from Italy and one from Chile, were assayed to obtain SOD IEPs. Italian populations had previously been identified at pathotype level by bioassays according to the generally accepted international test using different resistant potato cultivars and clones. The cluster analysis carried out on the SOD IEPs of all the populations tested formed four distinct groups within G. rostochiensis and only one within G. pallida. Pathotype identification of Globodera populations by SOD IEPs was not able to discriminate between bioassay standard couples Ro2/Ro3, Ro4/Ro5 and Pa2/Pa3. Therefore, three groups were assigned to Ro1, Ro2/3 and Ro4/5, and a fourth group to Pa2/Pa3. Four Venezuelan populations, not identified at pathotype level by bioassays, formed a distinct fifth group. By means of the method described herein, four additional unknown Venezuelan populations could be assigned to Ro1 group and one to Ro2/Ro3 group; one G. pallida population from Chile was assigned to Pa2/Pa3 group.