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Sound production in Cyprinodon bifasciatus, a pupfish endemic to the Cuatro Ciénegas basin, Coahuila, México, is documented. The average dominant frequency is 409 Hz, with an average duration of 55 msec. Calls were produced by males during pursuits of conspecifics and cichlids, during territory patrol, while following a female and after spawning. There are differences in call structure between contexts, especially postspawn calls versus pursuit and courtship calls. There may be differences between sites, especially Mojarral Oeste versus Becerra and Churince. Given sound production in this species of Cyprinodon and the related genus Fundulus, it is possible that sound production is more widespread than previously thought in Atherinimorpha. In addition, this is another documentation of sound production in a clear freshwater species indicating that sound production is widespread in clear freshwater fish.