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Abstract Background Animals living at high altitude must adapt to environments with hypoxia and low temperatures, but relatively little is known about underlying genetic changes. Toad-headed lizards of the genus Phrynocephalus cover a broad altitudinal gradient of over 4000 m and are useful models for studies of such adaptive responses. In one of the first studies to have considered selection on mitochondrial protein-coding regions in an ectothermic group distributed over such a wide range of environments, we analysed nineteen complete mitochondrial genomes from all Chinese Phrynocephalus (including eight genomes sequenced for the first time). Initial analyses used site and branch-site model (program: PAML) approaches to examine nonsynonymous: synonymous substitution rates across the mtDNA tree. Results Ten positively selected sites were discovered, nine of which corresponded to subunits ND2, ND3, ND4, ND5, and ND6 within the respiratory chain enzyme mitochondrial Complex I (NADH Coenzyme Q oxidoreductase). Four of these sites showed evidence of general long-term selection across the group while the remainder showed evidence of episodic selection across different branches of the tree. Some of these branches corresponded to increases in altitude and/or latitude. Analyses of physicochemical changes in protein structures revealed that residue changes at sites that were under selection corresponded to major functional differences. Analyses of coevolution point to coevolution of selected sites within the ND4 subunit, with key sites associated with proton translocation across the mitochondrial membrane. Conclusions Our results identify mitochondrial Complex I as a target for environment-mediated selection in this group of lizards, a complex that frequently appears to be under selection in other organisms. This makes these lizards good candidates for more detailed future studies of molecular evolution.