SummaryRoot‐knot nematodes (RKNs) are infamous plant pathogens in tomato production, causing considerable losses in agriculture worldwide.Mi‐1is the only commercially available RKN‐resistance gene; however, the resistance is inactivated when the soil temperature is over 28 °C.Mi‐9in wild tomato (Solanum arcanumLA2157) has stable resistance to RKNs under high temperature but has not been cloned and applied. In this study, a chromosome‐scale genome assembly ofS. arcanumLA2157 was constructed through Nanopore and Hi‐C sequencing. Based on molecular markers ofMi‐9and comparative genomic analysis, the localization region and candidateMi‐9genes cluster consisting of seven nucleotide‐binding sites and leucine‐rich repeat (NBS‐LRR) genes were located. Transcriptional expression profiles confirmed that five of the seven candidate genes were expressed in root tissue. Moreover, virus‐induced gene silencing of theSarc_034200gene resulted in increased susceptibility ofS. arcanumLA2157 toMeloidogyne incognita, and genetic transformation of theSarc_034200gene in susceptibleSolanum pimpinellifoliumconferred significant resistance toM. incognitaat 25 °C and 30 °C and showed hypersensitive responses at nematode infection sites. This suggested thatSarc_034200is theMi‐9gene. In summary, we cloned, confirmed and applied the heat‐stable RKN‐resistance geneMi‐9, which is of great significance to tomato breeding for nematode resistance.