The sonic hedgehog (Shh) pathway plays indispensable roles in the morphogenesis of mouse epithelial linings of the oral cavity and respiratory and digestive tubes. However, no enhancers that regulate regional Shh expression within the epithelial linings have been identified so far. In this study, comparison of genomic sequences across mammalian species and teleost fishes revealed three novel conserved non-coding sequences (CNCSs) that cluster in a region 600 to 900 kb upstream of the transcriptional start site of the mouse Shh gene. These CNCSs drive regional transgenic lacZ reporter expression in the epithelial lining of the oral cavity, pharynx, lung and gut. Together, these enhancers recapitulate the endogenous Shh expression domain within the major epithelial linings. Notably, genomic arrangement of the three CNCSs shows co-linearity that mirrors the order of the epithelial expression domains along the anteroposterior body axis. The results suggest that the three CNCSs are epithelial lining-specific long-range Shh enhancers, and that their actions partition the continuous epithelial linings into three domains: ectoderm-derived oral cavity, endoderm-derived pharynx, and respiratory and digestive tubes of the mouse. Targeted deletion of the pharyngeal epithelium specific CNCS results in loss of endogenous Shh expression in the pharynx and postnatal lethality owing to hypoplasia of the soft palate, epiglottis and arytenoid. Thus, this long-range enhancer is indispensable for morphogenesis of the pharyngeal apparatus.