Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance toFusarium oxysporumf. sp.Cubense(Foc), the causal agent of this disease, is genetically dissected in this study using twoMusa acuminatassp.Malaccensissegregating populations, segregating forFocTropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of ‘DH-Pahang’ reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent ‘Ma850’ and a susceptible line ‘Ma848’, to show that the STR4 resistance co-segregated with marker ‘28820’ at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as ‘Pahang’, ‘SH-3362’, ‘SH-3217’, ‘Ma-ITC0250’, and ‘DH-Pahang/CIRAD 930’. Additional screening in the International Institute for Tropical Agriculture’s collection suggests that the dominant allele is common among the elite ‘Matooke’ NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.