Cluster of differentiation 133 (CD133) is recognized as a stem cell marker for normal and cancerous tissues. Using cell culture and real‑time fluorescent polymerase chain reaction, CD133 expression was analyzed in osteosarcoma tissue and Saos‑2 cell lines. In addition, cancer stem cell‑related gene expression in the Saos‑2 cell line was determined to explore the mechanisms underlying tumorigenesis and high drug resistance in osteosarcoma. CD133+ cells were found to be widely distributed in various types of osteosarcoma tissue. Following cell culture, cells entered the G2/M and S cell cycle stages from G0/G1. Levels of CD133+ cells decreased to normal levels rapidly over the course of cell culture. Colony forming efficiency was higher in the CD133+ compared with the CD133‑ subpopulation of Saos‑2 cells. Expression levels of stem cell‑related genes, including multidrug resistance protein 1 (MDR1) and sex determining region Y‑box 2 (Sox2) in the CD133+ subpopulation of cells were found to be significantly higher compared with the CD133‑ subpopulation. These observations indicate that CD133+ Saos‑2 cells exhibit stem cell characteristics, including low abundance, quiescence and a high potential to undergo differentiation, as well as expression of key stem cell regulatory and drug resistance genes, which may cause osteosarcoma and high drug resistance.