Genomic data can improve our understanding on the phylogenetic relationship among Tibetan highland fishes. The whole mitochondrial genome of Gymnocypris eckloni generated in this study is 16,784 bp in length, containing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes, and one non-coding control region (D-Loop). Phylogenetic analysis recovers a non-monophyetic population of G. eckloni in the Qaidam basin, representing two distinct lineages designated ‘Qiadam A' and ‘Qaidam B', within Qaidam A clustering with Chuanchia labiosa and Schizopygopisis pylzovi captured in the Yellow River and Qaidam B grouping with G. eckloni from the Yellow River. Our research may helpful to further reconsideration of clearer taxonomy and improvement of biodiversity conservation strategy of Tibetan highland fishes.