Brain tumor-initiating cells (BTICs) are self-renewing multipotent cells critical for tumor maintenance and growth. Using single-cell microfluidic profiling, we identified multiple subpopulations of BTICs coexisting in human glioblastoma, characterized by distinct surface marker expression and single-cell molecular profiles relating to divergent bulk tissue molecular subtypes. These data suggest BTIC subpopulation heterogeneity as an underlying source of intra-tumoral bulk tissue molecular heterogeneity, and will support future studies into BTIC subpopulation-specific therapies. Stem Cells 2016;34:1702-1707.