Glioma stem cells (GSCs) may be a source of tumor progression and recurrence after multimodal therapy, because of their high invasive potential. The purpose of this study was to compare the invasive and migratory properties of GSCs and non-GSCs and examine the distribution of these cells in a mouse xenograft model. Three GSC lines, G144, Y02, and Y10, cultured from human glioblastoma, were used in the study. Matrigel-invasion assays of infiltration and time-lapse studies of migration were performed for comparison of the GSCs with the corresponding differentiated non-GSC lines. Cells were also transplanted into mouse brain and the different distribution of GSCs and non-GSCs was examined in the tumor xenograft model. All 3 GSC lines had greater invasion and migration ability than the corresponding non-GSCs. In vivo, GSCs infiltrated more widely than non-GSCs and reached the contralateral hemisphere via the corpus callosum in the early stage of tumorigenesis. GSCs also primarily penetrated the subventricular zone (SVZ). GSCs have high invasive potential and tend to be present in the outer tumor bulk and infiltrate the contralateral hemisphere via the corpus callosum, in addition to penetrating the SVZ.