The dispersal abilities and the population genetic structure of nematodes living in the soil are poorly known. In the present study, we have pursued these issues in the potato cyst nematode Globodera pallida, which parasitizes potato roots and is indigenous to South America. A hierarchical sampling regime was conducted in Peru to investigate gene flow on regional, field and plant scales. Multilocus genotypes of single individuals were obtained using eight polymorphic microsatellites markers. Large heterozygote deficiencies were observed at most loci. The limited active dispersal of larvae from their cyst, which favours mating between (half) siblings, could be responsible for this pattern. Within fields, as well as among fields within regions (even 35 km apart), low FST values suggest extensive gene flow. Among fields within regions, only 1.5–4.4% genetic variability was observed. Passive dispersal of cysts by natural means (wind, running water, or wild animals) or by anthropogenic means (tillage, movement of infected seed tubers) is probably responsible for the results observed. Among regions, high FST values were observed. Thus long-range dispersal (more than 320 km apart) is probably limited by major biogeographical barriers such as the mountains found in the Andean Cordillera. These results provide useful information for the management of resistant varieties, to slow down the emergence and spread of resistance-breaking pathotypes.