Childhood rhabdomyosarcoma (RMS) has two major histological subtypes: alveolar (aRMS) and embryonal. The aim of the study was to monitor minimal disseminated disease (MDD) using real-time quantitative reverse-transcription PCR (RQ-RT-PCR) of the PAX3-FKHR, PAX7-FKHR fusion genes and myoD1 gene. We prepared an assay using RQ-RT-PCR for a quantitative assessment of MDD in aRMS by using hydrolysis probe for quantification of PAX3-FKHR, PAX7-FKHR and myoD1 genes and beta-2-microglobulin housekeeping gene. Primary tumor samples (44), samples of local recurrences (26) from 48 patients with aRMS were examined by nested RT-PCR and RQ-RT-PCR techniques. Additionally, bone marrow samples (115), peripheral blood progenitor cell samples (27), and peripheral blood samples (25) from 33 aRMS patients were tested. PAX3/7-FKHR and myoD1 transcripts proved to be a sensitive tool for detection of MDD in RMS. We were able to identify 15/25 patients with bone marrow (BM) involvement at the time of presentation using RQ-RT-PCR. We analyzed PAX3-FKHR or PAX7-FKHR expression during the course of the disease. The RQ-RT-PCR results correlated well with nested RT-PCR results (p < 0.0001). The presence of metastases is the most adverse prognostic factor in RMS, and bone marrow is a frequent site of the tumor dissemination in RMS, especially in aRMS. Our results detecting the fusion transcripts or myoD1 transcript in the BM or peripheral blood suggest that patients with positive findings are at high risk of the tumor progression.