Netrin1 is a diffusible factor that attracts commissural axons to the floor plate of the spinal cord. Recent evidence indicates that Netrin1 is widely expressed and functions in the development of multiple organ systems. In mammals, there are three genes encoding Netrins, whereas in zebrafish, only the Netrin1 orthologs netrin1a and netrin1b have been identified. Here, we have cloned two new zebrafish Netrins, netrin2 and netrin4, and present a comparative sequence and expression analysis. Despite significant sequence similarity with netrin1a/netrin1b, netrin2 displays a unique expression pattern. Netrin2 transcript is first detected in the notochord and in developing somites at early somitogenesis. By late somitogenesis, netrin2 is expressed in the fourth rhombomere and is subsequently expressed in the hindbrain and otic vesicles. In contrast, netrin4 is detected only at very low levels during early development. The nonoverlapping expression patterns of these four Netrins suggest that they may play unique roles in zebrafish development. Developmental Dynamics 234:726–731, 2005. © 2005 Wiley-Liss, Inc.