AbstractThe molecules that constitute myelin are critical for the integrity of axon/myelin‐units and thus speed and precision of impulse propagation. In the CNS, the protein composition of oligodendrocyte‐derived myelin has evolutionarily diverged and differs from that in the PNS. Here, we hypothesized that the CNS myelin proteome also displays variations within the same species. We thus used quantitative mass spectrometry to compare myelin purified from mouse brains at three developmental timepoints, from brains of male and female mice, and from four CNS regions. We find that most structural myelin proteins are of approximately similar abundance across all tested conditions. However, the abundance of multiple other proteins differs markedly over time, implying that the myelin proteome matures between P18 and P75 and then remains relatively constant until at least 6 months of age. Myelin maturation involves a decrease of cytoskeleton‐associated proteins involved in sheath growth and wrapping, along with an increase of all subunits of the septin filament that stabilizes mature myelin, and of multiple other proteins which potentially exert protective functions. Among the latter, quinoid dihydropteridine reductase (QDPR) emerges as a highly specific marker for mature oligodendrocytes and myelin. Conversely, female and male mice display essentially similar myelin proteomes. Across the four CNS regions analyzed, we note that spinal cord myelin exhibits a comparatively high abundance of HCN2‐channels, required for particularly long sheaths. These findings show that CNS myelination involves developmental maturation of myelin protein composition, and regional differences, but absence of evidence for sexual dimorphism.