Schizothoracinae is an endemic fish of the Tibetan Plateau and its surrounding areas. The phylogenetic and adaptive evolutionary mechanisms of this subfamily are unclear, and the taxonomic status of Gymnocypris chilianensis remains controversial. This study evaluated the complete mitochondrial genome sequences of 65 species of Schizothoracinae in 11 specified genera, with the aim of providing a genomic basis for elucidating the above issues. The corresponding results are as follows: (1) Phylogeny. The results showed that the Schizothoracinae subfamily is not monophyletic but belongs to two different clades, with each taxon containing several generic species. The primitive taxa of the Schizothoracinae fishes were grouped into one clade; the specialized taxa and highly specialized taxa were grouped into another clade; (2) Adaptive evolution. The results showed that no positive selection sites were detected at any of the 13 protein-coding genes with ω ratios ＜1. Positive selection sites were also found in Atp6, ND2, ND3, ND4, and ND5. Nonetheless, positive selection primarily occurred in respiratory chain complex I (NADH1-6); the mitochondrial respiratory chain is the most important source of biologically reactive oxygen species (ROS), which may explain the adaptive evolutionary mechanism of the subfamily Schizothoracinae fishes in high-altitude hypoxic environments. For the divergence time study, the divergence time of the primitive taxon Schizothoracinae fishes was estimated to be before 3.200 Ma; the specialized taxon Schizothoracinae fishes began to differentiate between 5.972 Ma and 5.320 Ma in the late Miocene period; (3) Taxonomic status of G. chilianensis. Based on the Kimura two-parameter model, the genetic distance between G. chilianensis and Schizopygopsis kessleri was 0.0015. Nonetheless, the genetic distance between G. chilianensis and Gymnocypris eckloni was 0.0673 (＞0.0600), indicating species differentiation. Overall, it is clear that, at the molecular level, G. chilianensis is a distinct species of the genus Gymnocypris and not a subspecies of G. chilianensis. Therefore, this study provided evidence to resolve the long-standing disagreement over the taxonomic status of G. chilianensis.