The Manyara tilapia (Oreochromis amphimelas), native to the Saline Rift Valley Lakes (SRVLs) in north‐central Tanzania, has recently experienced population declines mainly due to overfishing and severe droughts. This, combined with the introduction of Nile tilapia (O. niloticus), which poses a risk of hybridisation, could potentially erode essential genetic traits necessary for their survival. To address these concerns, our study analysed partial cytochrome oxidase subunit I sequences (627 base pairs) from 139 Manyara tilapia and 79 Nile tilapia specimens collected from the SRVLs to test two hypotheses: (i) whether there is no transfer of mitochondrial DNA between these species, and (ii) whether Manyara tilapia populations in the SRVLs have remained genetically connected despite reported declines. The results revealed six distinct haplotypes for Manyara tilapia and three for Nile tilapia. These haplotypes formed separate clusters for each species, with no shared or closely related haplotypes, indicating limited or no transfer of mitochondrial DNA between them. Additionally, we found evidence of restricted genetic connectivity among Manyara tilapia populations in the SRVLs, with populations in Lakes Manyara and Eyasi displaying limited genetic exchange among themselves and with those in other SRVLs. Our findings highlight the importance of sustained conservation efforts in protected areas around Lake Manyara and Lake Eyasi to safeguard the unique populations of Manyara tilapia in these lakes. However, limited connectivity with other SRVLs suggests that conservation initiatives in these areas may have a limited impact on these lakes. Thus, future efforts should prioritise stocks in Lakes Kindai, Singida and Sulunga, focusing on protecting fish refugia, particularly the rivers connected to these lakes, which serve as critical habitats during the dry season and as important reservoirs for replenishing these populations.