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The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg -expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut. [Display omitted] • Adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. • Larval Wg ring in the anterior zone of hindgut was not the precursors of adult hindgut. • The progenitors of the adult pylorus and ileum were situated in the ci expressing cells in the larval anterior zone. • The progenitors of the adult rectum were located in the trunk region of pylorus including hh expressing cells. [ABSTRACT FROM AUTHOR]