Meloidogyne chitwoodi and M. fallax are temperate-adapted root-knot nematodes (RKN) with exceptionally wide host ranges, including potato, tomato and other high-value crops, to which they inflict severe yield and quality loss. Both species are regulated as quarantine organisms in Switzerland. Recent detections of these nematodes have raised concerns about their introduction, establishment, and spread within Swiss agriculture. This study provides the first integrated characterization of one M. chitwoodi and four M. fallax populations from Switzerland. A newly discovered M. chitwoodi population was classified as Race 1, Roza pathotype through differential host assays and molecular marker analysis. Genetic relationships were further assessed with a multilocus barcoding approach targeting nuclear rRNA (18 S, 28 S, ITS), mitochondrial genes (COI, COII), and the 5–18 S intergenic spacer. RKN population sequences were analyzed together with global reference datasets to infer phylogenetic placement and putative origins. Phylogenetic analyses confirmed the identity of all Swiss populations. Intraspecific resolution was consistently low across all barcoding loci, indicating that standard multilocus barcodes have limited power to distinguish among populations of M. chitwoodi and M. fallax. Despite this limitation, the results contribute to a better understanding of the genetic diversity of M. chitwoodi and M. fallax in Switzerland and their relationship to global populations, supporting future surveillance and management efforts. These findings establish a genetic baseline for Swiss RKN surveillance, facilitate tracing of future incursions, and support the design of more effective phytosanitary and management strategies aimed at containing these economically important pests.