BACKGROUND: Despite intense investigation, the ability to treat high-grade glioma (HGG) has remained dismal, in part due to molecular and phenotypic heterogeneity. The cancer stem cell, a cell believed to be the clonogenic core of tumors including human glioma, has been a major focus for the development of new therapies but emerging evidence has exposed the dynamic and heterogenous characteristics of this stem-like population that facilitates evasion from current therapies. METHODS: To capture and target the complexity and heterogeneity of glioma, we employed a combinatorial phage-display biopanning strategy to isolate peptides that specifically bind and home in vivo to key disease reservoirs within glioma; namely the invasive and stem-like populations of glioma cells (GSC). Synthetic peptides, individually or in combination, were conjugated to gadolinium or a chemotherapeutic agent and administered to animals bearing orthotopic tumors established from human glioma-like-stem cells. RESULTS: Using this approach we identified a panel of peptides with the ability to detect the heterogeneity of patient gliomas in vivo successfully imaging a range of tumors including diffuse infiltrating tumours that are otherwise invisible by conventional imaging technologies. In addition, we identified peptides with multifunctional capabilities including peptides that functionally block glioma invasion, define a subpopulation of GSCs, and target chemotherapeutic agents in vivo. CONCLUSIONS: These data highlight the utility of the identified peptides as platforms for the precise and sensitive imaging of these heterogenous tumors and for the development of molecularly targeted therapeutics. SECONDARY CATEGORY: Tumor Biology.