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The aim of this study was to report a new locality for invasive mosquitofish Gambusia holbrooki in Iranian inland waters. In this study, the presence of G. holbrooki is reported in Kouski Spring located in the desert area of Yazd Province in the central plateau of Iran. Due to the special characteristics of invasive mosquitofish including small size, ability to spread rapidly, adaptability to different environments, extensive competition with other species, and success in rapid colonization, the distribution pattern of this species in inland waters is spreading rapidly. Since the introduction of exotic fish species into new ecosystems can have many ecological consequences, therefore, an integrated ecosystem management approach in the inland waters of Iran is crucial.