Abstract: The basic helix-loop-helix (bHLH) transcription factors are the products of main regulatory genes in metazoans. Regulatory genes are usually believed to play a large role in morphological and genetic diversifications and are often characterized by elevatedrates of evolution. In previous studies, it was showed there was much genetic diversification among the members of Hairy and Enhancer of split (Hairy/E(spl) or H/E(spl)) bHLH family. This promoted us to do further research on the mechanism of genetic diversification within the H/E(spl) bHLH family. With all the data available, we carried out an evolutionary analysis among sequence sites of the enlarged H/E(spl) bHLH family and reported that all the Hes gene lineages were under purifying selection, but the Hes2, Hes3, Hes5 lineages evolved faster than the other ones with relatively higher omega values. We found nine sites of Hes5 genes under significantly positive selection and concluded that the diversity of H/E(spl) genes has been established through relaxed selective constraint.