Middle Tennessee populations of the Streamside Salamander, Ambystoma barbouri, delimit the southern extent of the species' range and are geographically isolated from more northerly populations. Few populations have been discovered in Tennessee, and all of them are located in the Inner Nashville Basin ecological subregion of the Interior Plateau. We surveyed for breeding activity in first- and second-order streams in the southern Inner Nashville Basin to determine the species distribution and to examine the status of and existing threats to extant populations. Streamside salamanders were found at five of 40 localities in southern Rutherford, northern Bedford and northeastern Marshall County, and at only 4 of 6 previously known breeding sites. Continued habitat fragmentation and alteration in association with the urbanization of Rutherford County threaten existing A. barbouri populations, which may represent the last remaining populations in the state. We recommend state and local agencies develop a habitat conservation plan to preserve and improve first- and second-order breeding sites and the surrounding forests used by adult Streamside Salamanders.