While the molecular and cellular effects of opioids have been extensively studied, the precise mechanisms by which these drugs target specific brain regions over time remain unclear. Similarly, despite well-documented sex differences in opioid responses, the anatomical basis for this sexual dimorphism is not well characterized. To address these questions, we developed an automated, scalable, and unbiased approach for whole-brain anatomical mapping of the neuronal activity marker c-Fos in response to acute morphine exposure. Using ribbon scanning confocal microscopy, we imaged whole cleared brains from male and female wild-type mice at 1 hour and 4 hours post-morphine administration. Our whole-brain analysis of c-Fos expression revealed distinct patterns of morphine-induced regional brain activation across time and sex. Notably, we observed a greater number of structures with significant activity differences at 4 hours compared to 1 hour. In male mice, significant changes were primarily localized to regions within the dopamine system, whereas in female mice, they were concentrated in cortical regions. By combining high-throughput imaging with whole-brain expression analysis, particularly in the context of opioid actions, our approach provides a more comprehensive understanding of how drugs of abuse affect the brain.