Three-dimensional (3D) cultures are indispensable for capturing tumor heterogeneity in colorectal cancer (CRC) in vitro. Although 3D cultures (such as sphere-forming assay and organoid culture) can partially preserve the morphological and molecular characteristics of primary CRC, whether these 3D cultures maintain the long-term stemness of cancer stem cells (CSCs) remains largely unknown. In the present study, spheres and organoids were generated side by side using individual primary CRC specimens, then respectively processed as serial passages. The results revealed that during serial passages, the percentage of CSCs (such as cluster of differentiation-133(+) and Wnt(+) cells) in organoids and the tumor-initiating capacity of organoid-derived cells were constant, while they gradually increased in the sphere-derived cells. Furthermore, during serial passages, resistance to chemotherapeutic agents (including 5-fluorouracil and oxaliplatin) in sphere- and organoid-derived cells was evaluated. The results indicated that the percentage of chemoresistant cells was constant in serial organoid cultures; however, it gradually increased in the serial sphere-forming assays. Taken together, the results of the present study comprehensively demonstrate that, with regard to long-term culture in vitro, organoid culture may be useful in maintaining tumor heterogeneity and the levels of chemoresistant cells, while the sphere formation assay enriches for CSCs and chemoresistant cells.