We examined the expression patterns of 4 layer-specific genes in monkey and mouse cortices by fluorescence double in situ hybridization. Based on their coexpression profiles, we were able to distinguish several subpopulations of deep layer neurons. One group was characterized by the expression of ER81 and the lack of Nurr1 mRNAs and mainly localized to layer 5. In monkeys, this neuronal group was further subdivided by 5-HT2C receptor mRNA expression. The 5-HT2C⁺/ER81⁺ neurons were located in layer 5B in most cortical areas, but they intruded layer 6 in the primary visual area (V1). Another group of neurons, in monkey layer 6, was characterized by Nurr1 mRNA expression and was further subdivided as Nurr1⁺/connective tissue growth factor (CTGF)⁻ and Nurr1⁺/CTGF⁺ neurons in layers 6A and 6B, respectively. The Nurr1⁺/CTGF⁺ neurons coexpressed ER81 mRNA in monkeys but not in mice. On the basis of tracer injections in 3 monkeys, we found that the Nurr1⁺ neurons in layer 6A send some corticocortical, but not corticopulvinar, projections. Although the Nurr1⁺/CTGF⁻ neurons were restricted to lateral regions in the mouse cortex, they were present throughout the monkey cortex. Thus, an architectonic heterogeneity across areas and species was revealed for the neuronal subpopulations with distinct gene expression profiles.