Ewing sarcoma (ES) is an aggressive primary malignant bone tumor that predominantly affects children and young adults. Multimodal treatment approaches have markedly improved the survival of patients with localized ES. However, local recurrence and distant metastasis following curative therapies remain a main concern for patients with ES. Recent studies have suggested that slow‑cycling cells (SCCs) are associated with tumor progression, local recurrence and distant metastasis in various types of cancers. According to the results of these studies, it was hypothesized that SCCs may play a critical role in tumor progression, chemoresistance and local/distal recurrence in patients with ES. The present study applied a label‑retaining system using carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify and isolate SCCs in ES cell lines. In addition, the properties of SCCs, including sphere formation ability, cell cycle distribution and chemoresistance, in comparison with non‑SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non‑SCCs; the identified genes not only inhibited cell cycle progression, but also promoted the malignant properties of SCCs. On the whole, the present study successfully identified SCCs in ES cells through a label‑retaining system using CFSE. Moreover, to the best of our knowledge, the present study is the first to describe the characteristic properties of SCCs in ES. The findings of this study, if confirmed, may prove to be useful in elucidating the underlying molecular mechanisms and identifying effective therapeutic targets for ES.