Potato cyst nematode (PCN) is a major pest of potato crops. Marker-assisted selection of PCN-resistant cultivars is consequently a key objective of many potato breeding programmes. The genetic marker TG689, linked to the resistance gene H1, is the current marker of choice for the prediction of PCN resistance. Polymorphism at TG689 was evaluated across 373 cultivars from Australian and Scottish breeding programmes, revealing the loss of association between the predictive allele and trait locus. Two recently identified genetic markers flanking the H1 locus were then evaluated for association with PCN-resistant phenotypes across 73 cultivars. With a single exception, presence or absence of a 452-bp amplicon for the 57R marker was predictive for the target phenotype. Further evaluation of the 57R marker on more than 300 cultivars from the Australian and Scottish potato breeding programmes provided additional evidence for robust diagnostic capability. To enable routine implementation, the 57R marker was converted into a high-throughput, fluorochrome detectionbased assay and screened across a broader collection of parental cultivars and advanced breeding selections for the prediction of PCN resistance.