The reprogramming of cancer cells includes shifts in glucose and glycogen metabolism. The aim of our work was to check the ability of forming glycogen grains in hepatocellular tumor cell lines of various dedifferentiation levels. We studied the monolayer culture established in vitro after explanting cells from rat ascites Zajdela hepatoma strain C (ZH-C) as a "parental" line and its five daughter clonal sublines: the holoclonal sublines 3H, 5F, 6H and the meroclonal ones 1E, 9C, which possess, respectively, the properties of cancer stem-like cells (CSLCs) and cancer progenitor-like cells (CPLCs). Besides, we studied four permanent cell lines of a rat hepatoma HTC, two murine hepatomas BWTG3 and MH-22a, and human hepatoblastoma HepG2. We used normal rat hepatocytes as positive control cells that form glycogen. We estimated relative cell dedifferentiation levels of the studied lines via analysis of cell morphology, morphometry and motility character on stained cell preparations and lifetime video files. Glycogen in the cells was detected using a Schiff type Au-SO reagent. All studied hepatocellular tumor lines were not of equal dedifferentiation level as manifested by different nucleus-to-cytoplasm ratio, by epithelium-like or fibroblast-like morphology, by tight or loosen intercellular contacts, by cell migration of collective or individual types. Glycogen fluorescence of uneven intensity was observed in all normal rat hepatocytes, but only in some cell groups or in single cells of hepatocellular tumor lines. The large or small fluorescent grains were found not only in relatively less dedifferentiated parental ZH-C line, BWTG3 and HepG2 lines, but also in moderately dedifferentiated 1E and HTC lines and even in severely dedifferentiated 3H, 5F and 6H sublines, as well as in the islets of the rat ascites hepatoma induced in vivo by the injection of 3H cells (the tumor-initiating cells). On the other hand, MH-22 and 9C lines, being relatively less and moderately dedifferentiated, showed no glycogen fluorescence. Thus, in 10 tumor cell lines of hepatocellular origin, an ability to reserve glycogen manifested no obvious dependency on their dedifferentiation level. Glycogen grains were detected in some cells even of the severely dedifferentiated lines: in single CSLCs of holoclonal ZH sublines grown in vitro and in a majority of tumor-initiating cells derived from ascites hepatoma in vivo. We suggest that dynamic changes in glycogen formation in CSLCs and tumor-initiating cells might be of importance for their dedifferentiation, self-renewal in vitro, survival and metastasis in vivo. The role of glycogen in maintaining viability and metastasis of tumor cells is to be further studied.