AylacostomaSpix, 1827, contains species that are the subject of focused conservation efforts under the auspices of the 'AylacostomaProject', the only ex situ conservation programme for freshwater gastropods in South America. Two species from the High Paraná River (Argentina-Paraguay) are included in this programme (Aylacostoma chloroticumHylton Scott, 1954 and Aylacostoma brunneumVogler & Peso, 2014), as their habitats have disappeared as a consequence of the filling of the Yacyretá Reservoir in the 1990s. At present, A.chloroticum is restricted to only one known wild population in a small and fragile habitat, and wild populations of A.brunneum are presumed to have gone extinct. We used partial sequences of the cytochrome oxidase subunit I gene to provide the first phylogeographical perspective on these species from a limited dataset containing representatives of several wild populations that are successfully being bred in captivity. We found low genetic diversity and two haplotypes in A.chloroticum, and absence of variation with one haplotype in A.brunneum. The reservoir's entry zone was identified to be of great interest for conservation, and is where we suggest re-introductions and translocations should be targeted, to preserve the future evolutionary potential of the extant diversity.