Previous studies have demonstrated some tumors develop or maintain a small sub-population of cells with stem cell-like properties. This Cancer Stem Cells (CSC) may exhibit differential properties that allow their escape from traditional radiation or chemotherapy treatments and may therefore be responsible for cancer recurrence. Few studies have explored this phenomenon among oral cancer cell lines, therefore the objective of this study was to examine multiple oral cancer cell lines to determine if any or all contained subpopulations of CSCs. Multiple commercially available Oral Squamous Carcinoma Cell (OSCC) lines were obtained for this study, including SCC15, SCC25 and CAL27. Cells were cultured for CSC screening and isolation. RNA was isolated from any potential CSC isolates for biomarker screening and verification. All OSCC lines examined developed adhesion-independent tumor spheres (AiTS), a characteristic phenotype of oral CSC. Each AiTS was manually isolated for separate, independent culture and analysis. RNA extracted from the AiTS revealed differential expression of specific CSC markers, including CD44, CD133, ABCG, CXCR6 and NANOG. These biomarkers were not observed in RNA extracted from the remaining non-CSC cell cultures. Although a previous study from this group successfully isolated AiTS from one cervical and one oral cancer cell line, this may be the first study to isolate CSC from multiple oral cancer cell lines and verify both cell-surface and intracellular CSC biomarkers. These results may suggest that many tumors and oral cancers could harbor AiTS and CSC and that screening for these sub-populations may provide guidance for treatment and therapy to improve oral cancer survival rates.