Fusarium oxysporum isolates collected from onions in the UK and other countries were characterized using sequences of the transfer elongation factor 1-α (TEF) gene and compared with published sequence data for 10 other isolates. Isolates associated with diseased onion bulbs in the UK formed two clades. Isolates from both clades were selected for pathogenicity testing and to develop a rapid seedling assay to screen commercial onion cultivars for resistance to F. oxysporum f. sp. cepae (FOC), the cause of basal rot. Differences in the levels of aggressiveness between isolates were observed and isolates from both clades were pathogenic. Differences in resistance/susceptibility were also observed amongst 10 commercial onion cultivars, with cvs Ailsa Craig Prizewinner and White Lisbon showing the highest levels of resistance. The results from the seedling assay were supported by those from a subsequent onion bulb rot assay. Thus, this study reports the development of a rapid, simple and repeatable seedling assay that can be used to screen large numbers of onion cultivars for resistance to FOC and which is indicative of resistance at the bulb stage.