The Allen Institute Mouse Diversity Study characterizes gene expression across genetic backgrounds and sex, expanding beyond the adult male C57BL/6J reference brain comprising the Allen Mouse Brain Atlas to include seven strains of male mice and female C57BL/6J mice. This freely accessible online resource allows for simultaneous navigation and comparison of different mouse brains at high resolution, providing users with a dataset to probe for possible differences in gene regulation. A panel of 49 well known drug target genes that are expressed in the adult and developing brain and encompass sites of action for FDA-approved drugs were assayed for both the strain and sex components of the project. In addition, 12 other genes predicted to show variability across specimens were selected for each component and assayed. Gene expression was detected using colorimetric RNA in situ hybridization (ISH), providing cellular level anatomic resolution.