It would be very useful to be able to classify brain tumor stem cells (BTSCs) by certain criteria to afford the design of specific or individualized treatment. Here, we studied two BTSC lines with differing biological and molecular features and whose respective features were well preserved after cryopreservation as single cells in SFM or 90% serum with 10% DMSO, a method not previously reported. The resuscitated BTSCs shared properties indistinguishable from their respective parental cells, including tumor sphere forming potentials, growth and differentiation properties, and tumorigenesis in vivo. The two cell lines also had differing molecule profiles, which can be well preserved after cryopreservation, similar to that of their respective primary tumors. Therefore, BTSCs from different patients, that have their own properties, were well retained by the present cryopreservation method, which might be a useful and reliable method for preserving BTSCs for long-term studies, such as classification and specific therapy design.