The development of ef?cient and repeatable protocols for biobanking and prolonged storage of cancer stem cells (CSCs), with minimum alterations in biological function, is valuable and desired, particularly for retrospective analysis and clinical applications. In particular, data regarding the effect of cryopreservation on CSCs's functional features is scarce. In this regard, few studies have been shown that 3D spheroid structures, which enriched for CSCs, can keep their biological phenotype and genetic profiles. Here, for the first time, we present data on cryopreservation of CT-26 colonospheres, with the focus on essential stem cell-like properties after thawing. Tumor biopsy-derived colonospheres were frozen in standard freezing media (90% fetal bovine serum + 10% dimethyl sulfoxide) and stored in liquid nitrogen for 10 months. Then, cryopreservation effect on preservation of CSCs-related features was verified using real-time polymerase chain reaction for evaluation of stemness genes and flow cytometry for the putative colorectal CSC surface biomarkers. The self-renewal capacity of thawed spheres was also compared with their fresh counterparts using serial formation assay. Finally, tumorigenic capacity of both groups was evaluated in immunocompetence mouse model. Our data indicated that postthawed colonospheres had high viability without drastic alteration in biological and structural features and maintained self-renewal potential after sequential passages. Real-time analysis showed that both fresh and frozen colonospheres displayed similar expression pattern for key stemness genes: SOX2 and OCT4. Cryopreserved spheroids expressed CD133, CD166, and DCLK1 CSCs surface biomarkers at elevated levels when compared with parental as non-cryopreserved counterparts. Our electron scanning microscopy micrographs clearly demonstrated that postthawed colonospheres retain their integrity and cell surface morphology and characteristics. We also found that both fresh and frozen spheroids were equally tumorigenic. This study represented an effective strategy for reliable storage of intact CT-26 colonospheres; this can provide researchers with a functionally reliable repository of murine colorectal CSCs for their future CSCs projects.