We have conducted a screen to identify developmentally regulated enhancers that drive tissue-specific Gal4 expression in zebrafish. We obtained 63 stable transgenic lines with expression patterns in embryonic or adult zebrafish. The use of a newly identified minimal promoter from the medaka edar locus resulted in a relatively unbiased set of expression patterns representing many tissue types derived from all germ layers. Subsequent detailed characterization of selected lines showed strong and reproducible Gal4-driven GFP expression in diverse tissues, including neurons from the central and peripheral nervous systems, pigment cells, erythrocytes, and peridermal cells. By screening adults for GFP expression, we also isolated lines expressed in tissues of the adult zebrafish, including scales, fin rays, and joints. The new and efficient minimal promoter and large number of transactivating driver-lines we identified will provide the zebrafish community with a useful resource for further enhancer trap screening, as well as precise investigation of tissue-specific processes in vivo.