To enable quantitative high-content phenotyping in zebrafish using large in situ RNA probe libraries, we have developed an optical projection tomography platform capable of imaging complex anatomical structures and gene expression patterns rapidly and affordably in three dimensions and at high resolution. In contrast to standard 3D imaging modalities--which require expensive hardware, are often not amenable to high-throughput sample handling, and are restricted to fluorescent readouts--our system can be assembled using commercially available components and enables 3D imaging of gene expression patterns at cellular resolution in non-embedded zebrafish larvae using standard chromogenic in situ protocols. Image acquisition time is less than 7 seconds per embryo and the system can be paired with automated fluidics such as the Vertebrate Automated Screening Technology (VAST) system we previously developed for higher-throughput larvae handling. We have additionally developed algorithms to automate all aspects of 3D reconstruction and registration to stage-specific anatomical atlases, allowing for rapid visualization of morphology and gene expression patterns throughout early development. By applying correlation analysis to a diverse library of brain-specific probes, we are able to quantify subtle changes in gene expression patterns between experimental groups throughout the entire brains of intact animals. Applying this approach to the well-characterized tof/fezf2 mutant, we are not only able to automatically extract virtually all previously described early developmental defects, but also to detect novel aspects of the phenotype. Quantitative phenotypic data sets such as these allow labs to more easily compare phenome-level observations and will be an invaluable tool for analyzing the numerous mutant lines being generated through the rapid adoption of genome editing tools.