Cancer stem cells (CSCs) or cancer stem cell-like cells (CSC-LCs) are a minority population of cells that relate to tumor progression, metastasis and drug resistance. To identify CSC-LCs in oral squamous cell carcinoma (OSCC), we used two OSCC cell lines, SAS and OSC20, and cell surface markers, CD44v3 and CD24. In addition, we examined CD44v3 and CD24 expression immunohistochemically and evaluated the relationship between the expression and clinicopathological parameters in 50 OSCC tissues. In SAS and OSC20, CD44v3+/CD24- cells showed a higher sphere forming ability than the other fractions, i.e., CD44v3+/CD24+, CD44v3-/CD24- and CD44v3-/CD24+ cells. The proportion of CD44v3+/CD24- cells in SAS and OSC20 was 10.7 and 24.1%, respectively. Regarding SAS, CD44v3+/CD24- cells also showed a higher drug resistance for CDDP, 5-FU and cetuximab and expressed higher mRNA levels of CSC property-related genes than the other cell fractions. The tumorigenicity of CD44v3+/CD24- cells was not significantly different from the other fractions in SAS. An immunohistochemical study revealed a significant correlation between CD44v3 expression in the invasive portion and lymph node metastasis. Kaplan Meier analysis revealed cases with CD44v3 expression in the invasive portion tended to show poor overall survival (OS) compared with those without CD44v3, and there was a significant difference in OS between CD44v3+/CD24- and CD44v3-/CD24- immunophenotypes in the invasive portion. In conclusion, the results suggest that the CD44v3+/CD24- cell population displays CSC-LC properties in a human OSCC cell line. Additionally, we present evidence that CD44v3 immunoexpression and CD44v3+/CD24- immunophenotypes could give prognostic information associated with unfavorable clinical outcomes.