Identifying the structural basis of developmental anomalies using imaging, gross morphology, and histology has led to new insights into gene function. In the last 5 years, the DTCC Consortium, one of three members of the NIH KOMP2 project and a part of the IMPC, has generated knockout mouse lines for 850 unique genes selected for having little to no pre-existing functional annotation. Mutant lines that survive post-weaning are tested in a multi-system phenotyping pipeline from 9 to 16 weeks of age, while lines that fail to give birth to homozygous offspring or survive post-weaning at 3 weeks of age undergo an alternative pipeline. To identify developmental abnormalities leading to death before (embryo lethal) and after (subviable) birth, we screened mutants for viability at different developmental stages to determine the window of lethality and imaged the animals using sensitive methods for each stage. Our screen uses optical projection tomography (OPT) for embryos identified as homozygous lethal at embryonic day (E)12.5 but viable at E9.5, micro-computed tomography (micro-CT) for lines viable at E15.5 but lethal before birth, and high resolution magnetic resonance imaging (MRI) of whole brains of postnatal day (P)7 mice for lines that are viable at birth but lethal before weaning. DTCC uses an automated image analysis system that is applied to 8 biological replicates comprised of either sex per mutant line. A mutant population average scan is overlaid for comparison to an age-matched wild type population average 3D atlas to identify and quantitate differences with <5% false discovery rate. Automation eliminates bias, increases throughput, and enhances sensitivity to detect differences. Our analysis also includes evaluation and annotation of gross morphology in freshly dissected embryos using a structured set of e-Mouse Atlas Project (EMAP) and Mammalian Phenotype (MP) ontology terms. Staining for lacZ expression analysis is completed at E12.5 for all embryo lethal and neonate subviable lines that have been analyzed at E15.5. Histological assessment of paraffin-embedded sections is done on embryos and placentas for lines analyzed at E9.5 or E15.5 and on brains and whole bodies of neonates analyzed at P7. Examples of pleiotropic, quantified, and characterized gross and subtle phenotypes from each of the imaging modalities and histology will be presented. To date, our pre-weaning viability screen of 688 mutant lines has found 197 lines (29%) either lethal (114 lines; 17%) or subviable (83 lines; 12%). These morphological findings from image analysis with histology of mutant embryos and neonatal mice have provided new and informative insights into gene function essential for mammalian development. Supported by NIH grants U42OD011175 and U54HG006364.