The validity of the mitochondrial cytochrome C oxidase subunit I (CO I) sequences from Schizothoracids in GenBank as a molecular marker was determined in this study. Multiple sequence alignment, divergence from reference sequence assessment, and phylogenetic reconstruction were employed to evaluate the sequence homology, species diagnosis, and phylogenetic resolution of CO I sequences from Schizothoracids in GenBank. The primary alignment result for 1431 downloaded sequences revealed three sequences with significantly large indels; however, their homologous status was questionable. BLAST search and sequence information in GenBank confirmed that these sequences were Schizothoracid CO I sequences that were submitted as complements. Accordingly, prior to alignment, complement transition of these sequences should be carried out. Complete multiple sequence alignment revealed that all Schizothoracid CO I fragments in the GenBank nucleotide database were from near the 5? end to the middle of this coding gene. To retain more variation information, 35 short sequences were eliminated from the dataset. As a result, 1,396 sequences with 527 bp were analyzed, and 228 CO I haplotypes were defined. Intra-specific sharing haplotypes were common in species with multiple available sequences. However, 41 inter-specific sharing haplotypes were shared by more than one species. The average pairwise p-distance within the Schizothoracid CO I haplotypes was 9.5%, which is comparable with the cogeneric species level in fish. The average pairwise p-distances within the primitive, specialized, and highly specialized Schizothoracids were even shorter, a character of recent radiation that may reflect a short speciation history mainly driven by the elevation of the Qinghai-Tibet Plateau. Specimen misidentification was found in some Schizothoracid CO I sequences in GenBank, suggesting confirmation steps before use, such as comparison with reference sequences or inference from the phylogenetic reconstruction. Generally, the CO I sequence is a good molecular marker for Schizothoracids identification and primary evolutionary analysis, and the phylogenetic relationships among the three evolutionary grades of Schizothoracids are well resolved using this marker. Integrative analyses, including morphology, ecology, mitochondrial and nuclear DNA diversity, and phylogeny, would aid in taxonomic resolution and further investigation of hybridization and speciation in Schizothoracids.