The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying (TA) cells, respectively. This physical separation of stem and TA cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection (LCM), we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from 8 mice revealed a set of ~100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semi-quantitative RT-PCR confirmed the up-regulation of 3 limbal and 3 corneal genes. LacZ identification of epiregulin from epiregulin-null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated exclusively with the limbal epithelial basal cells. Within the limbal and corneal basal cells we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their “natural” quiescent state.