The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.