Effective methods for high-throughput screening and morphometric analysis are crucial for phenotyping the increasing number of mouse mutants that are being generated. Automated segmentation propagation for embryo phenotyping is an emerging application that enables noninvasive and rapid quantification of substructure volumetric data for morphometric analysis. We present a study to assess and validate the accuracy of brain and kidney volumes generated via segmentation propagation in an ex vivo mouse embryo MRI atlas comprising three different groups against the current “gold standard”—manual segmentation. Morphometric assessment showed good agreement between automatically and manually segmented volumes, demonstrating that it is possible to assess volumes for phenotyping a population of embryos using segmentation propagation with the same variation as manual segmentation. As part of this study, we have made our average atlas and segmented volumes freely available to the community for use in mouse embryo phenotyping studies. These MRI datasets and automated methods of analyses will be essential for meeting the challenge of high-throughput, automated embryo phenotyping. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.