BACKGROUND: Glioblastoma (GBM) is a cancer comprised of morphologically, genetically and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. METHODS: We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. RESULTS: Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide (TMZ) resistant, indicating that resistance to conventional GBM therapy preexists in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were identified with clone-specific drug screening. Genomic analyses identified genes and pathways that associate with specific functional behavior of single clones. In particular, increased expression and signaling of the autocrine/paracrine γ-aminobutyric acid (GABA) receptor GABRA3 correlates with TMZ sensitivity and patient outcome. CONCLUSIONS: Our results suggest that functional clonal profiling used to identify tumorigenic and drug resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies. SECONDARY CATEGORY: n/a.