Injury and inflammation in the gastric epithelium can cause disruption of the pathways that guide the differentiation of cell lineages, which in turn can cause persistent alterations in differentiation patterns, known as metaplasia. Metaplasia that occurs in the stomach is associated with increased risk for cancer. Methods for isolating distinct gastric epithelial cell populations would facilitate dissection of the molecular and cellular pathways that guide normal and metaplastic differentiation. Here, we identify alanyl aminopeptidase (CD13) as a specific surface marker of zymogenic chief cells (ZCs) in the gastric epithelium. We show that 1) among gastric epithelial cells alanyl aminopeptidase expression is confined to mature ZCs, and 2) its expression is lost en route to metaplasia in both mouse and human stomachs. With this new marker coupled with new techniques that we introduce for dissociating gastric epithelial cells and overcoming their constitutive autofluorescence, we are able to reliably isolate enriched populations of ZCs for both molecular analysis and for the establishment of ZC-derived ex vivo gastroid cultures.