We performed high-throughput mass spectrometry at high spatial resolution from individual regions (anterior cingulate and primary motor, somatosensory, and visual cortices) and layers of the neocortex (layers III, IV, and V) and cerebellum (granule cell layer), as well as the caudate nucleus in humans and chimpanzees. A total of 39 mass spectrometry peaks were matched with probable protein identifications in both species, allowing for comparison in expression. We explored how the pattern of protein expression varies across regions and cortical layers to provide insights into the differences in molecular phenotype of these neural structures between species. The expression of proteins differed principally in a region- and layer-specific pattern, with more subtle differences between species. Specifically, human and chimpanzee brains were similar in their distribution of proteins related to the regulation of transcription and enzyme activity but differed in their expression of proteins supporting aerobic metabolism. Whereas most work assessing molecular expression differences in the brains of primates has been performed on gene transcripts, this dataset extends current understanding of the differential molecular expression that may underlie human cognitive specializations.