The repulsive guidance molecule (RGMa) is involved in controlling the topography of retinal ganglion cell axons along the anterioposterior axis of the tectum. Here, we generated a new RGMa-monoclonal antibody and show that it is expressed in the developing retina, suggesting that it may regulate retinal axon pathfinding. We tested this hypothesis by using in ovo electroporation to either overexpress or downregulate RGMa in the eye. Anterograde labeling of retinal axons entering the optic tecta revealed abnormal phenotypes when RGMa expression is perturbed. These included the absence of terminal zone, the premature stalling of arborization of fibers, overshooting of terminal zone, aberrant axonal turns in the optic tectum and abnormal projections into deeper tectal layers. Moreover, RGMa overexpression frequently leads to intraretinal pathfinding errors. Thus, these data suggest that RGMa expression on retinal axons is a major determinant of topographic targeting in the retino-tectal projection and in the retina.