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High-energy freshwater environments such as rapids and waterfalls in the Upper-Paraná Atlantic Forest are home to highly endemic minute freshwater snails of the genus Acrorbis. Only one species, Acrorbis petricola, is currently included within this genus, whose geographical distribution is restricted to three known populations, one in Brazil and the other two in Argentina. Because of habitat specificity and limited geographical distribution, the species is considered vulnerable in Argentina and endangered in Brazil. In this work, we identify five new populations of A. petricola in southern Upper-Paraná Atlantic Forest, exclusively found on waterfalls from the Misiones Province, Argentina. Based on these populations and on specimens of one of the two historical populations from the Misiones Province, we explored the morphological features of shells and reproductive system of specimens from each location and provide the first molecular data on the species. We used DNA sequences from cytochrome c oxidase subunit I (COI) and 16S-rRNA genes to investigate the molecular diversity, genetic distances and genealogical relationships among populations. We verified the existence of intra- and interpopulation morphological variability, with the greatest variation being found in spire, spiral sculpture, penis sheath, flagella, prostatic diverticula and bursa copulatrix. We found interpopulation genetic diversity, with no intrapopulation variation, and identified six geographically structured genetic lineages with maximum genetic distances of up to 2.3%. Different combinations of morphological characters with the same genetic background within each locality were observed. The finding of new populations genetically differentiated not only broadens the known distribution of the species, but also illustrates that waterfall environments in the Atlantic Forest harbour a hidden diversity of Acrorbis that still remains to be discovered. This scenario suggests a complex evolutionary history that needs to be unveiled and taken into account for future development of conservation strategies in this endemic genus. [ABSTRACT FROM AUTHOR]