Abstract: Fish of the superfamily Cobitoidea sensu stricto (namely loaches) exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene (MC1R) plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MC1R gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae+ (Cobitidae+(Balitoridae+Nemacheilidae)). Gene evolutionary analyses indicated that MC1R in loaches was under purifying selection pressure, with various sites having different d_N/d_S values. Both Botiidae and Cobitidae had lower d_N/d_S values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoridae and Nemacheilidae, both had larger d_N/d_S values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MC1R gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.