Among fish parasitic nematodes Rhabdochona is one of the most speciose genera, with c. 100 species. Twelve congeneric species occur in Mexican freshwater fishes, in a region located between the Nearctic and Neotropical biogeographical regions. Host association and biogeographical history have determined the high species richness of Rhabdochona in Mexico. One of these species, Rhabdochona mexicana, is highly specific to the characid genus Astyanax. Characids are a group of freshwater fish with Neotropical affinity. In this paper, we explore the genetic diversity of R. mexicana through samples obtained from populations of Astyanax spp. across river basins of Mexico and Guatemala. Sequences of one mitochondrial and two ribosomal genes were obtained from 38 individuals and analysed using Maximum Likelihood and Bayesian Inference analysis. Phylogenetic analyses using cox1, and a concatenated alignment of 18S + 28S + cox1 recovered two genetic lineages. One of them corresponded with R. mexicana sensu stricto; this lineage included three reciprocally monophyletic subgroups; the other lineage was highly divergent and represented a putative candidate species. A detailed morphological study was conducted to corroborate the molecular findings. We describe a new species herein and discuss the implications of using molecular tools to increase our knowledge about the diversity of a speciose genus such as Rhabdochona.