The mammalian auditory system comprises a complex network of brain regions. Interpretations and comparisons of experimental results from this system depend on appropriate anatomical identification of auditory structures. The Waxholm Space (WHS) atlas of the Sprague Dawley rat brain (Papp et al., Neuroimage 97:374-86, 2014) is an open access, three-dimensional reference atlas defined in an ex-vivo magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) volume. Version 2.0 of the atlas (Kjonigsen et al., Neuroimage 108:441-9, 2015) includes detailed delineations of the hippocampus and several major subcortical regions, but only few auditory structures. To amend this, we have delineated the complete ascending auditory system from the cochlea to the cerebral cortex. 40 new brain structure delineations have been added, and the delineations of 10 regions have been revised based on the interpretation of image features in the WHS rat brain MRI/DTI volumes. We here describe and validate the new delineations in relation to corresponding cell- and myelin-stained histological sections and previous literature. We found it possible to delineate all main regions and the majority of subregions and fibre tracts of the ascending auditory pathway, apart from the auditory cortex, for which delineations were extrapolated from a conventional two-dimensional atlas. By contrast, only parts of the descending pathways were discernible in the template. Version 3.0 of the atlas, with altogether 118 anatomical delineations, is shared via the NeuroImaging Tools and Resources Collaboratory (www.nitrc.org). Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.