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Vascular wilt caused by the ascomycete fungal pathogen <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> (<i>Foc</i>) is a major constraint of banana production around the world. The virulent race, namely Tropical Race 4, can infect all Cavendish-type banana plants and is now widespread across the globe, causing devastating losses to global banana production. In this study, we characterized <i>Foc</i> Subtropical Race 4 (STR4) resistance in a wild banana relative which, through estimated genome size and ancestry analysis, was confirmed to be <i>Musa acuminata</i> ssp. <i>malaccensis</i>. Using a self-derived F₂ population segregating for STR4 resistance, quantitative trait loci sequencing (QTL-seq) was performed on bulks consisting of resistant and susceptible individuals. Changes in SNP index between the bulks revealed a major QTL located on the distal end of the long arm of chromosome 3. Multiple resistance genes are present in this region. Identification of chromosome regions conferring resistance to <i>Foc</i> can facilitate marker assisted selection in breeding programs and paves the way towards identifying genes underpinning resistance.