PCR (conventional and real-time) and DNA sequence analysis were used to identify species and genotypes of potato cyst nematodes from sites in Victoria, Australia. Only Globodera rostochiensis was detected. Sequence analyses of these isolates of PCN have confirmed the PCR-based results and have revealed the presence of genetically diverse populations in infested fields. However, the sequence variation was not in the diagnostic primer binding sites. The melting peaks, from multiplex real-time PCR analysis, for Globodera pallida and G. rostochiensis were 83.3 and 88.7, respectively. The importance of DNA extraction, PCR and sequence analysis for the molecular identification of PCN is discussed. This study has significant implications for detecting species of PCN in order to monitor/develop control strategies for the PCN of quarantine importance.