SummaryThe microphthalmia‐associated transcription factor (MITF) is a member of the MYC family of basic helix–loop–helix leucine zipper transcription factors. The corresponding gene was initially discovered in the mouse based on mutations which affect the development of several different cell types, including melanocytes and retinal pigment epithelium cells. Subsequently, it was shown to be associated with deafness and hypo‐pigmentation disorders in humans. More recently, the gene has been shown to be critical in melanoma formation and to play a role in melanocyte stem cell maintenance. Thus, the mouse Mitf gene represents an important model system for the study of human disease as well as an interesting model for the study of transcription factor function in the organism. Here we use the evolutionary relationship of Mitf genes from numerous distantly related species, including vertebrates and invertebrates, to identify novel conserved domains in the Mitf protein and regions of possible functional importance in the 3′ untranslated region. We also characterize the nine different 5′ exons of the Mitf gene and identify a new 5′ exon in the Drosophila Mitf gene. Our analysis sheds new light on the conservation of the Mitf gene and protein and opens the door for further functional analysis.