Due to the detrimental effect of formaldehyde on DNA, ethanol has replaced formalin as the primary preservative for animal specimens. However, short-term formalin fixation of specimens might be applied during field collection. In an increasing number of studies, DNA extraction and sequencing have been successfully conducted from formalin-fixed specimens. Here the DNA from five specimens of Triplophysadalaica (Kessler, 1876) were extracted and performed high-throughput sequencing. Four of the specimens underwent short-term fixation with formalin and were subsequently transferred to ethanol. One was continuously stored in ethanol. No significant difference of DNA quality and amount were observed among these samples. Followed by assembly and annotation, five mitochondrial genomes ranging in length from 16,569 to 16,572 bp were obtained. Additionally, previously published data of other individuals or species were included to perform phylogenetic analyses. In the reconstructed trees, all eight individuals of T.dalaica form a monophyletic group within the Triplophysa branch. The group is divided into three clades: (1) samples from the Yellow River, (2) those from the Yangtze River, and (3) those from the Haihe River, and the Lake Dali Nur. This study sheds initial light on the phylogeographic relationships among different populations of T.dalaica , and will support the research about its evolutionary history in the future.