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Divergence in acoustic signals plays an important role in the production and maintenance of biodiversity in numerous taxa. In this study, we assess patterns of acoustic divergence in geographically isolated and sympatric subspecies of barn swallows (Hirundo rustica), including analyses of whether song differentiation varies with geographical isolation, genetic distance and climatic distance. We provide the first description of geographical variation in song among five of six currently recognized barn swallow subspecies. Temporal traits describing terminal trills were the most distinct song traits among subspecies, adding to growing evidence that trills are important in speciation among many birds, insects and fish. Across a ~6000 km transect of Russia, acoustic distance was predicted by genetic and geographical distance, but not climatic distance. We also found no reproductive character displacement of song traits in a contact zone between H. r. rustica and H. r. tytleri. Based on patterns discovered in this study, we infer an important role of sexual selection, genetic and/or cultural drift in the gradual build-up of acoustic divergence, which is accelerated in small populations. [ABSTRACT FROM AUTHOR]