Glioblastoma (GBM) is a highly aggressive brain cancer with limited treatments and poor patient survival. GBM tumors are heterogeneous containing a complex mixture of dividing cells, differentiated cells, and cancer stem cells. It is unclear, however, how these different cell populations contribute to tumor growth or whether they exhibit differential responses to chemotherapy. Here we set out to address these questions using a zebrafish xenograft transplant model (Welker et al., 2016). We found that a small population of differentiated vimentin positive tumor cells, but a majority of Sox2 positive putative cancer stem cells, were dividing during tumor growth. We also observed co-expression of Sox2 and GFAP, another suggested marker of glioma cancer stem cells, indicating that the putative cancer stem cells in GBM9 tumors expressed both of these markers. To determine how these different tumor cell populations responded to chemotherapy, we treated animals with temozolomide and assessed these cell populations immediately after treatment and 5 and 10 days after treatment cessation. As expected we found a significant decrease in dividing cells after treatment. We also found a significant decrease in vimentin positive cells, but not in Sox2 or GFAP positive cells. However, the Sox2 positive cells significantly increased 5 days after TMZ treatment. These data support that putative glioma cancer stem cells are more resistant to TMZ treatment and may contribute to tumor regrowth after chemotherapy.