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Behaviorally relevant sex differences are often associated with structural differences in the brain and many diseases are sexually dimorphic in prevalence and progression. Characterizing sex differences is imperative to gaining a complete understanding of behavior and disease which will, in turn, allow for a balanced approach to scientific research and the development of therapies. In this study, we generated novel tissue probability maps (TPMs) based on 30 male and 30 female in vivo C57BL/6 mouse brain magnetic resonance images and used voxel-based morphometry (VBM) to analyze sex differences. Females displayed larger anterior hippocampus, basolateral amygdala, and lateral cerebellar cortex volumes, while males exhibited larger cerebral cortex, medial amygdala, and medial cerebellar cortex volumes. Atlas-based morphometry (ABM) revealed a statistically significant sex difference in cortical volume and no difference in whole cerebellar volume. This validated our VBM findings that showed a larger cerebral cortex in male mice and a pattern of dimorphism in the cerebellum where the lateral portion was larger in females and the medial portion was larger in males. These results are consonant with previous ex vivo studies examining sex differences, but also suggest further regions of interest.Voxel-based morphometry analysis of sex differences in the in vivo C57BL/6J brain.Male mice demonstrated larger cerebral cortices, medial cerebella, and posterior hippocampi.Female mice demonstrated large lateral cerebella and anterior hippocampi.Results were remarkably consonant with previous studies.