Stem decay and root rot of Amaranthus hybridus, caused by Fusarium oxysporumis a serious threat to the commercial production of this crop in South Africa. Five Amaranthus hybridusvarieties were examined in vitrofor sensitivity to a culture filtrate of Fusarium oxysporum.The phytotoxicity of the culture filtrate was assessed for its inhibitory effect on callus and seeding root growth, as well as on the viability of callus cells. The five varieties exhibited a significant amount of variation in response to the culture filtrate of the pathogen. Variety 17 was the most sensitive variety in each bioassay, whereas variety 20 displayed least sensitivity to the culture filtrate. Callus of variety 20 grew well in the presence of concentrations of culture filtrate that were toxic to another four varieties and the percentage mortality of callus cells after exposure to the filtrate was also the lowest of the five varieties. Root growth of variety 20 was also least affected by exposure to the culture filtrate for up to 6 days. To our knowledge, the presence of resistance in germplasm of A. hybridusto culture filtrates of F. oxysporumhas not been demonstrated before. The present study is therefore of significant value to breeding programs aimed at speedily finding amaranth cultivars that are resistant to this important pathogen.