Purpose: Currently, the two most influential gastric stem cell marker candidates are CD44 and CD133. The aim of this study was to make a comparison and determine the appropriate marker for use in gastric cancer stem cell research. Materials and Methods: We analyzed the expressions of CD44, CD133, and CD24 from the gastric cancer cell lines MKN45, MKN74, KATO-III, NCI-N87, SNU-1, SNU-216, SNU-601, SNU-638, and SNU-688 using flow cytometry. In addition, we measured the change in viability after applying 5 fluorouracil (5-FU) to the MKN45, MKN74, KATO-III, and NCI-N87 cell lines using a Cell Counting Kit 8. Results: CD133 expression was above moderate in the KATO-III, SNU-216, SNU-601 cell lines, whereas it was below 1% in the remaining cell lines. CD44 was expressed at levels above 5% in all gastric cancer cell lines. The effect of 5-FU on viability and CD133 or CD44 expression in the cell lines were not related. Conclusions: Expression of CD133 positive cells was insufficient in the gastric cancer cell lines. Therefore, of the cell lines tested, CD44 was the most appropriate tumor maker for research on gastric cancer stem cells.