Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. To achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 was used to label the cancer cells of a pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery or single-color FGS could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant-cancer surgery.