PURPOSE: To characterize the microarchitecture of anterior limbal stroma in healthy individuals using in vivo confocal microscopy (IVCM) and to correlate it with mesenchymal stem cells (MSCs), a component of the limbal niche. METHODS: The corneal side of the superior limbus was scanned in 30 eyes of 17 normal subjects beyond the basal epithelium, deep into the stroma using an HRT III laser scanning microscope. The IVCM findings were correlated with the immunohistochemical features of MSCs in the anterior limbal stroma. RESULTS: Clusters of hyperreflective structures were observed in the anterior limbal stroma, subjacent to the basal epithelium (depth, 50.2 ± 8.7 μm to 98 ± 12.8 μm), but not in the corneal stroma. The structures showed unique morphology compared with epithelial cells, keratocytes, neurons, and dendritic cells. In parallel, confocal analysis of immunostained sections showed clusters of cells, double positive for MSC-specific markers (CD90 and CD105) in the anterior limbal stroma at a depth of 55.3 ± 12.7 μm to 72 ± 37.6 μm. The organization and distribution of the MSC clusters locates them within the hyperreflective region in the anterior limbal stroma. CONCLUSIONS: The hyperreflective structures, demonstrated for the first time in the human anterior limbal stroma, probably represent an important component of the limbal niche. Our approach of in vivo imaging may pave the way for assessing the limbal stromal health.