Colorectal cancer (CRC) is a major cause of cancer-related mortality in the world. Recently, a number of studies have demonstrated that cancer stem cells (CSCs) present in colorectal cancer tissues, are responsible for resistance to conventional therapies. Therefore, effective recognition of CSCs is of great importance. In the present study, to explore the potential characterizations of CSCs by the expression of specific cell surface markers such as CD133 and CD44, we screened six CRC cell lines using western blotting, immunofluorescence and flow cytometry. SW620, one of the six cell lines analyzed, was sorted into four subpopulations by fluorescence activated cell sorting (FACS). The capability of colony formation, proliferation rate, apoptosis, drug resistance, as well as their migratory and invasion potential were detected. The results revealed that the combination of CD44 and CD133 correlates with the features of CSCs in SW620 cells. CD44 positive cells showed more robust colony formation, higher proliferation, less spontaneous apoptosis, a higher resistance to drug-induced cell death, and were enriched after drug treatment. Among CD44 positive SW620 cells, the CD133 negative subpopulation was more migratory and invasive, which means that CD44+CD133- correlates with most of features proposed for CSCs. Overall, the data presented herein showed that CRCs have a wide range of expression for CD44 and CD133; it is unlikely the CSCs can be characterized by any single marker or the same set of markers for all colon cancer cells. For SW620 cells, the CSCs are likely represented by the CD44+CD133- surface marker. This finding of CSC markers represented by one positive and one negative is in line with CSCs in other tumors, such as CD34+CD38- for acute myeloid leukemia; CD44+CD24- for breast and pancreatic tumors. The absence of surface molecule(s) on CSCs will make it even more difficult to track and target this group of minority cells.