The Cre/loxP system has been used extensively in mouse models with a limitation of one lineage at a time. Differences in function and other properties among populations of adult β-cells is termed β-cell heterogeneity, which was recently associated with diabetic phenotypes. Nevertheless, the presence of a developmentally derived β-cell heterogeneity is unclear. Here, we have developed a novel dual lineage-tracing technology, using a combination of two recombinase systems, Dre/RoxP and Cre/LoxP, to independently trace green fluorescent Pdx1-lineage cells and red fluorescent Ptf1alineage cells in the developing and adult mouse pancreas. We detected a few Pdx1 + /Ptf1a − lineage cells in addition to the vast majority of Pdx1 + /Ptf1a + lineage cells in the pancreas. Moreover, Pdx1 + /Ptf1a + lineage β-cells had fewer Ki-67 + proliferating β-cells, and expressed higher mRNA levels of insulin, Glut2, Pdx1, MafA and Nkx6.1, but lower CCND1 and CDK4 levels, compared with Pdx1 + / Ptf1a − lineage β-cells. Furthermore, more TSQ-high, SSC-high cells were detected in the Pdx1 + Ptf1a + lineage population than in the Pdx1 + Ptf1a − lineage population. Together, these data suggest that differential activation of Ptf1a in the developing pancreas may correlate with this β-cell heterogeneity.