Recent studies in distinct glioma types have separately identified phenotypically distinct populations of tumor propagating cells (TPCs). Enrichment of TPCs by cell surface marker expression (CD133 [PMID:17051156], NG2 [PMID: 21907924]) has revealed the possibility of multiple TPC populations, within a single tumor, that have distinct properties and therapy sensitivities. Here we identify and compare the coexisting populations of glioblastoma multiforme (GBM) cells expressing the stem marker CD133 and the progenitor marker NG2. We compare the proliferative properties of CD133+ and NG2+ cells, and investigate how polarity and mode of division may determine other functional properties including cell cycle regulation and therapy sensitivity. Established and primary GBM cell cultures were analyzed by immunofluorescence and flow cytometry for expression of CD133 and NG2, alongside markers of polarity and proliferation. We showed that CD133+ and NG2+ GBM cells coexist as predominantly separate subpopulations. CD133+ cells are highly polarized and, compared to NG2+ or marker-negative subpopulations, are slower dividing. Cell cycle and gene expression analyses showed that the increase in cell cycle length in CD133+ cells is due to an extended G2-M period. As expected CD133+ cells have increased expression of DNA-damage response genes, however paradoxically we also detected increased activation of polo-like kinase 1 (PLK1). CD133+ cells were uniquely sensitive to the PLK1 inhibitor BI2536 in vivo, and treatment of intracranial GBM xenografts with the BRAF inhibitor PLX4720 and BI2536 showed greater anti-tumor activity than either alone. Our studies identified two putative TPC subpopulations in GBM with distinct cell cycle dynamics, DNA damage response and cell division mode. Ongoing work will investigate the role of Plk1 activity in linking these divergent phenotypes. Understanding this phenotypic heterogeneity in adjacent TPC types will allow the design of treatment strategies that eradicate multiple putative TPC populations.