Artesian springs are particularly sensitive freshwater ecosystems, characterized by specialized flora and fauna which, owing to their isolation, are often endemic. Thus, protection of spring habitats and endemic spring biota is important for biodiversity conservation, particularly because human impacts such as invasive species, habitat destruction, and fragmentation can have such devastating effects. This study investigated the mitochondrial DNA (mtDNA) genetic diversity and structure of two critically endangered (IUCN) freshwater fish species: red-finned blue-eye Scaturiginichthys vermeilipinnis, and Edgbaston goby Chlamydogobius squamigenus, with the aim of assisting management agencies to establish relocated populations that conserve the genetic integrity and evolutionary potential of both species. 2862 bp from multiple mtDNA genes were amplified from 111 red-finned blue-eye individuals (17 from the 1990s and 94 from 2010), and a 660 bp segment of mtDNA cytochrome b was amplified from 111 Edgbaston goby individuals (from 2010). In total, 14 concatenated mtDNA haplotypes were identified in red-finned blue-eye and three cytb haplotypes were identified in Edgbaston goby. Overall assessment of the genetic diversity and structure of the endemic fish fauna from the Edgbaston springs revealed low levels of diversity in both species and a loss of haplotypes in red-finned blue-eye over time. In addition, both species displayed significant genetic structure: at the spring scale for red-finned blue-eye and at the spring group scale for Edgbaston goby. It is concluded that without the appropriate management of relocated populations, such as considering genetic structure and diversity and selecting high quality habitats, these species are at a severe risk of losing evolutionary potential and becoming extinct. This study provides a valuable example for conservation managers of the contribution that population genetic studies can make regarding the adaptive management of endangered species.