The family Hemisinidae consists exclusively of freshwater snails and has long been understudied, only recently recognized as a distinct evolutionary lineage among limnic cerithioideans. Most South American species of this family belong to the genus Aylacostoma, with limited information available beyond their original descriptions. Aylacostoma chloroticum, a threatened freshwater snail endemic to the High Paraná River between Argentina and Paraguay, is currently part of an ex-situ conservation program due to habitat alterations caused by the Yacyretá Binational Reservoir. Molecular data on this species are scarce, with only one prior study addressing the genetic composition of captive populations. In this study, next-generation sequencing was used to sequence the complete mitochondrial genome of A. chloroticum, marking the first mitogenome contribution for both Aylacostoma and Hemisinidae. The mitogenome is 15,740 bp long and contains the typical 37 genes found in animal mitogenomes, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Comparative analyses revealed a conserved gene order, high A+T content, and negative AT and GC skews, consistent with other Cerithioidea species. Secondary structure models for ribosomal and transfer RNAs were also generated, providing the first complete models for Aylacostoma and Hemisinidae. Phylogenetic analyses, based on protein-coding genes and complementary analyses using 16S-rRNA and 28S-rRNA genes, confirmed the monophyly of Hemisinidae and its close evolutionary relationship with the families Paludomidae and Thiaridae. This research enhances the understanding of the mitochondrial architecture of Cerithioidea and provides new insights into the evolutionary relationships of Neotropical hemisinids.
