Root-knot nematodes (RKN; Meloidogyne spp.) are predominant polyphagous pests of crops in the Central Valley of Chile. Twenty RKN populations from this region were collected from diverse crops and subsequently identified with both sequence-characterized amplified region and isoenzyme markers. Populations included three RKN species: Meloidogyne ethiopica (75%), M. javanica (15%), and M. arenaria (10%). This is the first report of the high prevalence and wide host range of M. ethiopica in Chile. The host status of three Prunus rootstocks for isolates obtained from the Chilean RKN populations was then evaluated. Rootstocks assessed included the peach rootstock Nemaguard and the plum rootstock Marianna 2624, both previously considered resistant, and the peach rootstock Pomona as a susceptible accession. In the first experiment, rootstocks were inoculated individually in pots with 10,000 second-stage, juveniles and eggs of each isolate, and reproduction and galling were evaluated 5 months after inoculation. In the second experiment, the six most aggressive RKN isolates were used in a mixture to evaluate the host response of the same three rootstocks. No RKN were detected on Marianna 2624 in both experiments, which confirmed its immune host status to M. arenaria and M. javanica; this is the first report of immunity to M. ethiopica. Even though Pomona was classified overall as susceptible, the response of this rootstock to the RKN isolates was highly variable and ranged from susceptible to resistant, depending on the RKN isolates. Nemaguard ranged from resistant (to each of the most aggressive isolates and to their mixture) to highly resistant (to M. arenaria isolates). Our results illustrate that Prunus rootstocks express different levels of resistance to RKN species. RKN resistance may be active either at the isolate level (as in Pomona), at the species level (as in Nemaguard toward M. javanica and M. arenaria), or at the genus level (as in Marianna 2624).