The dermis contains two distinct layers: the papillary and the reticular layers. In vitro cultures of the fibroblasts from these layers show that they are different. However, no molecular markers to differentiate between the two subtypes of fibroblasts are known. We performed gene expression analysis on cultured fibroblasts isolated from the papillary and reticular dermis. In all, 116 genes were found to be expressed differentially. Of these, 13 were validated by quantitative reverse transcriptase–PCR analysis and two markers could be validated at the protein level in monolayer cultures. Three markers showed differential expression in in vivo skin sections. The identified, characteristic markers of the two fibroblast subpopulations provide useful tools to perform functional studies on reticular and papillary fibroblasts.