SummaryRoot‐knot nematodes (RKNs) are infamous plant pathogens in tomato production, causing considerable losses in agriculture worldwide. Mi‐1 is the only commercially available RKN‐resistance gene; however, the resistance is inactivated when the soil temperature is over 28 °C. Mi‐9 in wild tomato (Solanum arcanum LA2157) has stable resistance to RKNs under high temperature but has not been cloned and applied. In this study, a chromosome‐scale genome assembly of S. arcanum LA2157 was constructed through Nanopore and Hi‐C sequencing. Based on molecular markers of Mi‐9 and comparative genomic analysis, the localization region and candidate Mi‐9 genes 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 the Sarc_034200 gene resulted in increased susceptibility of S. arcanum LA2157 to Meloidogyne incognita, and genetic transformation of the Sarc_034200 gene in susceptible Solanum pimpinellifolium conferred significant resistance to M. incognita at 25 °C and 30 °C and showed hypersensitive responses at nematode infection sites. This suggested that Sarc_034200 is the Mi‐9 gene. In summary, we cloned, confirmed and applied the heat‐stable RKN‐resistance gene Mi‐9, which is of great significance to tomato breeding for nematode resistance.