In the version of this article initially published, the Acknowledgements section was incomplete and has now been amended to include the following: “NIH BRAIN Initiative awards U01 MH121282 to J.R.E and M.M.B, U19 MH114831 to J.R.E. and E.M.C., U19 MH114830 to H.Z., U01 MH114819 to G.F., 1U01MH114828 to K.Z. and J.C., RF1MH123220 to M.H. and R.H.S., and U19 MH114821. NIH awards R01DC019370 to R.H., R24MH114815 to R.H. and O.R.W., and R24 MH114788 to O.R.W. Nancy and Buster Alvord Endowment to C.D.K.” The changes have been made to the HTML and PDF versions of the article.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals¹. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.