A rare subpopulation of cancer cells known as cancer stem cells (CSCs) have distinct characteristics resembling stem cells, including cell renewal capability, differentiation into multiple lineages, and endless proliferation potential. Cumulating evidence has revealed that CSCs are responsible for tumorigenicity, invasion, metastasis, and therapeutic resistance. Despite continued investigation of CSCs, in vivo behavior of CSCs is not yet fully understood. The in vivo imaging modalities of optical, nuclear, and magnetic resonance are currently being employed to investigate the complexity behind the CSCs behavior. Valuable information that were previously obscured by the limitations of in vitro techniques now are currently being revealed. These studies give us a more comprehensive insight about what happen to CSCs in vivo. This review will briefly discuss the recent findings on CSCs behavior as informed by in vivo imaging studies.