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The use of bioacoustics as a tool for bat research is rapidly increasing worldwide. There is substantial evidence that environmental factors such as weather conditions or habitat structure can affect echolocation call structure in bats and thus compromise proper species identification. However, intraspecific differences in echolocation due to geographical variation are poorly understood, which poses a number of issues in terms of method standardization. We examined acoustic data for Pteronotus cf. rubiginosus from the Central Amazon and the Guiana Shield. We provide the first evidence of intraspecific geographic variation in bat echolocation in the Neotropics, with calls significantly differing in almost all standard acoustic parameters for the two lineages of this clade. We complement our bioacoustic data with molecular and morphological data for both species. Considerable overlap in trait values prevents reliable …