The RMia gene, which confers resistance (R) to the root-knot nematodes (RKN) Meloidogyne incognita and Meloidogyne arenaria, has been shown to segregate in the peach rootstocks Nemared, Shalil, and Juseitou on LG2 of the Prunus map. Here, we report the high-resolution mapping of RMia in Nemared, using the peach genome sequence and 790 individuals from two segregating peach populations, the F2 crossMontclar x Nemared and the four-way cross [(Pamirskij × Rubira) × (Montclar × Nemared)], in which Montclar, Pamirskij, and Rubira are susceptible (S) to RKN. Among the simple sequence repeat (SSR) markers designed for an initial flanking region of more than 1 Mb, five SSR markers specific for Nemared were characterized. The genotyping and phenotyping of recombinant individuals in this interval narrowed the gene’s location to a 300 kb physical distance between the SSR markers AMPP117 and AMPP116. In this interval, SNP polymorphisms were recovered from 1-kb-sequenced DNA fragments that were selected at 20 kb intervals. Two SNP markers (A20SNP and SNP_APP91) were shown to flank the gene in a final 92-kb region, containing four candidate genes from the TIR–NBS– LRR family. Finally, we studied the polymorphism of three closely linked markers, SNP_APP92, SNP_APP91, and AMPP117, on 28 R or S accessions fromdiverse Prunus species or hybrids. These markers discriminated between most R and S accessions, suggesting that at least the R sources of Nemared, Nemaguard, and Shalil share a common resistant ancestor