Potato cyst nematodes (PCN) are damaging soilborne quarantine pests of potato in many parts of the world. There are two recognized species, Globodera pallida and G. rostochiensis, with only the latter species—the golden cyst nematode—present in Australia. PCN was first discovered in Australia in 1986 in Western Australia, where it was subsequently eradicated and area freedom for market access was reinstated. In Victoria, PCN was first detected in 1991 east of Melbourne. Since then, it has been found in a small number of localized regions to the south and east. Strict quarantine controls have been in place since each new detection. It has previously been speculated that there were multiple separate introductions of PCN into Victoria. Our study utilized a historic (years 2001 to 2014) PCN cyst reference collection to examine 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 eggs within cysts and screened using nine previously described polymorphic microsatellite markers in two multiplex polymerase chain reaction assays. Sequence variation of the internal transcribed spacer region of the DNA was also assessed and compared with previously published data. A hierarchical sampling strategy was used, comparing variability of larvae within cysts, within paddocks, and between local regions. This sampling revealed very little differentiation between Victorian populations, which share the same microsatellite allelic variation, with differences between local regions probably reflecting changes in allele frequencies over time. Our molecular assessment supports a probable single localized introduction into Victoria followed by limited spread to nearby areas. The Australian PCN examined appear genetically distinct from populations previously sampled worldwide; thus, any new exotic incursions, potentially bringing in additional PCN pathotypes, should be easily differentiated from existing established local PCN populations.