Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Diagnosis of RMS can be difficult when it appears as a small round-cell tumor without evidence of differentiation. Recently, a set of regulatory proteins expressed during skeletal muscle development has been described. Among them, MyoD1 has been detected by Northern blot and immunohistochemical analyses in normal skeletal muscle and RMS. Given the relevance of this marker in the diagnosis of RMS, we developed an assay to evaluate the expression of MyoD1 mRNA in small tissue specimens by reverse transcription polymerase chain reaction. Specificity and sensitivity of the assay was determined in a series of 25 tumor cell lines and 39 pediatric tumor samples, including 35 RMSs. Subsequently, we studied the expression of MyoD1 in bone marrow and peripheral blood stem cell specimens. We detected the MyoD1 transcript in normal skeletal muscle and in almost all RMSs, whereas no expression was found in non-RMS samples or in normal hematopoietic tissues. This assay showed high sensitivity and specificity, and it could be a useful molecular tool for the diagnosis of RMS within small roundcell tumors of childhood and for the detection of minimal bone marrow and peripheral blood stem cell involvement in children with RMS, regardless of the histological subtype.