The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are major pests of ware and seed potato (Solanum tuberosum L.) crops worldwide and severely impact the movement of potatoes around the globe through quarantine restrictions. In Australia, only G. rostochiensis has been discovered, on four separate occasions between 1986 and 2008. The infested areas are the subject of strict regulation and quarantine procedures and while they are considered to be contained, managing nematode populations remains a priority. This study has identified the G. rostochiensis Ro1 resistance-status of potato cultivars currently grown by Australian potato growers, and new cultivars emerging from the Australian Potato Breeding Program. Resistance was assessed by a simple and robust procedure carried out in a purpose-built quarantine facility. Of the 24 potato cultivars grown in the affected Koo Wee Rup district in 2004, 10 were resistant to nematode infestation, including the locally important cultivar Atlantic. Other cultivars important to the Victorian and Australian potato industry, such as Kennebec, Desiree, Sebago and Coliban, were classified as susceptible. Importantly, this study provided evidence that the Koo Wee Rup PCN population was able to complete its lifecycle on the native plant species, S. aviculare (kangaroo apple), potentially acting as an alternate host and spreading PCN among potato crops.