The recent development of genetic methods allows the delineation of species boundaries, especially in organisms where morphological characters are not reliable to differentiate species. However, few empirical studies have used these tools to delineate species among parasitic metazoans. Here we investigate the species boundaries of Clinostomum, a cosmopolitan trematode genus with complex life cycle. We sequenced a mitochondrial [cytochrome c oxidase subunit I (COI)] gene for multiple individuals (adults and metacercariae) from Middle-America. Bayesian phylogenetic analysis of the COI uncovered five reciprocally monophyletic clades. COI sequences were then explored using the Automatic Barcode Gap Discovery to identify putative species; this species delimitation method recognized six species. A subsample was sequenced for a nuclear gene (ITS1, 5·8S, ITS2), and a concatenated phylogenetic analysis was performed through Bayesian inference. The species delimitation of Middle-American Clinostomum was finally validated using a multispecies coalescent analysis (species tree). In total, five putative species are recognized among our samples. Mapping the second intermediate hosts (fish) onto the species tree suggests that metacercariae of these five species exhibit some level of host specificity towards their fish intermediate host (at the family level), irrespective of geographical distribution.