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Topographic organization of neurons is a hallmark of brain structure. The establishment of the connections between topographically organized brain regions has attracted much experimental attention, and it is widely accepted that molecular cues guide outgrowing axons to their targets in order to construct topographic maps. In a number of systems afferent axons are organized topographically along their trajectory as well, and it has been suggested that this pre- target sorting contributes to map formation. Neurons in auditory regions of the brain are arranged according to their best frequency (BF), the sound frequency they respond to optimally. This BF changes predictably with position along the so- called tonotopic axis. In the avian auditory brainstem, the tonotopic organization of the second- and third- order auditory neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) has been well described. In this study we examine whether the decussating NM axons forming the crossed dorsal cochlear tract (XDCT) and innervating the contralateral NL are arranged in a systematic manner. We electroporated dye into cells in different frequency regions of NM to anterogradely label their axons in XDCT. The placement of dye in NM was compared to the location of labeled axons in XDCT. Our results show that NM axons in XDCT are organized in a precise tonotopic manner along the rostrocaudal axis, spanning the entire rostrocaudal extent of both the origin and target nuclei. We propose that in the avian auditory brainstem, this pretarget axon sorting contributes to tonotopic map formation in NL. J. Comp. Neurol. 521:2310– 2320, 2013. © 2012 Wiley Periodicals, Inc.Combining multicell and single- cell labeling with a whole- brainstem imaging technique, the authors show topographic axon arrangement in the avian auditory brainstem. This pre- target axon sorting might contribute to tonotopic map formation in the auditory system.