Background: Rhabdomyosarcoma (RMS) is characterized by features of skeletal muscle and is comprised of two major histological subtypes, embryonal (E-RMS), and alveolar (A-RMS). Subsets of each RMS subtype demonstrate resistance to multimodal therapy leading to treatment failure. Cancer stem cells or cancer-initiating cells (CIC) represent a theorized population of cells that give rise to tumors and are responsible for treatment resistance. Procedure: We investigated the ability of CD133, a putative CIC marker, to distinguish a chemoresistant, myogenically primitive population in alveolar (RH30), and embryonal (RD) RMS cell lines. We tested CD133+/- cells for sensitivity to engineered herpes simplex virus (oHSV). Results: Relative to CD133- cells, CD133+ A-RMS, and E-RMS cells demonstrate an enhanced colony-forming ability, are less differentiated myogenically, and are more resistant to cytotoxic chemotherapy but equally sensitive to oHSV oncolysis. Compared to CD133- RD cells, CD133+ cells express relatively high levels of genes typically expressed in skeletal muscle progenitor satellite cells including PAX7, c-MET, and the GLI effectors of the hedgehog signaling pathway. In contrast, CD133+ RH30 cells were not associated with enhanced expression of satellite cell markers or Hh targets. Conclusions: Our findings demonstrate that CD133+ cells from A-RMS and E-RMS cell lines are characterized by a myogenically primitive phenotype. These cells have the capacity to form colonies in vitro and are more resistant to chemotherapy than CD133- cells. CD133 expression may denote a subset of RMS cells with an important role in tumorigenesis and treatment failure. These resistant cells may be effectively targeted by oHSV therapy. © 2012 Wiley Periodicals, Inc.