Potato cyst nematodes (PCN) are damaging soil-borne quarantine pests of potatoes in many parts of the world. There are two recognised species, Globodera pallida and G. rostochiensis, of which only G. rostochiensis is present in Australia. PCN was first discovered in Australia in 1986 in a suburb of Perth, Western Australia, was subsequently eradicated and area freedom reinstated. In Victoria, PCN was first detected in February 1991 in Wandin, east of Melbourne. Since then it has been found in small pockets in Gembrook (1991), Emerald, Keysborough and Boneo (1992), Kooweerup/Cora Lynn (2003) and Thorpdale (2008), also east of Melbourne. Strict quarantine controls have been in place since each detection. In 2007, it was speculated that there may have been up to 7 separate introductions of PCN into Victoria. In this study, we utilised the PCN cyst reference collection held by Crop Health Services, Agriculture Victoria, to examine the genetic variability of Victorian PCN populations to investigate potential historical origins and subsequent changes in the populations that might inform patterns of spread. DNA was extracted from single larvae dissected from cysts and screened using nine polymorphic microsatellite markers in two multiplex PCR assays, and the ITS region. A hierarchical sampling strategy was used, comparing variability of larvae (265) within cysts (54), within paddocks (33) and between regions (4). Considerable variation was detected in the Victorian populations and the results will be presented at the Conference.