Abstract We present the complete mitochondrial genome of Tzotzilthelphusa villarosalensisVillalobos & Álvarez, 2013, a freshwater crab from Mexico. This is the first mitochondrial genome reported for the superfamily Pseudothelphusoidea. We performed a de novo assembly from genome skimming reads. The mitochondrial genome of T. villarosalensis has a length of 17,203 bp and its gene topology is similar to other freshwater and marine families, e.g., the position of the cytochrome oxidase complex subunits and ATA synthetase, even the order of nad3, nad5, nad4, nad4L, nad6, and cytb. By contrast, the order and organization of genes like the 16S, 12S, and nad1 are different depending on the phylogenetic group with which they are compared. The phylogenetic analyses placed T. villarosalensis as a sister group of all freshwater crabs reported until now and as part of the subsection Heterotremata. This complete mitochondrial genome could be a reference for further genomic studies of the family Pseudothelphusidae and other primary freshwater crabs.