Mammalian esophagus exhibits a remarkable change in epithelial structure during the tran- sition from embryo to adult. However, the molecular mechanisms of esophageal epithelial development are not well understood. Zebrafish (Danio rerio), a common model organism for vertebrate development and gene function, has not previously been characterized as a model system for esophageal epithelial development. In this study, we characterized a piece of non-keratinized stratified squamous epithelium similar to human esophageal epi- thelium in the upper digestive tract of developing zebrafish. Under the microscope, this piece was detectable at 5dpf and became stratified at 7dpf. Expression of esophageal epi- thelial marker genes (Krt5, P63, Sox2 and Pax9) was detected by immunohistochemistry and in situ hybridization. Knockdown of P63, a gene known to be critical for esophageal epi- thelium, disrupted the development of this epithelium. With this model system, we found that Pax9 knockdown resulted in loss or disorganization of the squamous epithelium, as well as down-regulation of the differentiation markers Krt4 and Krt5. In summary, we charac- terized a region of stratified squamous epithelium in the zebrafish upper digestive tract which can be used for functional studies of candidate genes involved in esophageal epithe- lial biology.