Chromosomal translocations of t(2;13)(q35;q14) and t(1;13)(p36;q14), resulting in PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR) gene fusions, have been found to be specific molecular markers for alveolar rhabdomyosarcomas (ARMS) and can be identified in approximately 80% cases. As the prognosis of ARMS is worse than that of embryonal rhabdomyosarcomas (ERMS), it is important to accurately distinguish between these 2 subtypes. This distinction may be difficult on the basis of morphology alone. To detect the genetic alterations, reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color dual-fusion fluorescence in situ hybridization (FISH) have been used in most studies so far. In this study, we used FOXO1 (FKHR) gene break-apart FISH probe, which can detect both of the translocations involving the FOXO1 gene, and tested 20 cases of rhabdomyosarcoma (RMS) including 6 cases of ARMS, 8 ERMS, 1 pleomorphic type, 5 not otherwise specified (RMS-NOS), and 10 non-RMS sarcomas. A home-brew RT-PCR that could detect both PAX3-FOXO1 and PAX7-FOXO1 was also performed. Four pathologists independently reviewed all RMS and a consensus diagnosis was also reached in discrepant cases. Histologic and molecular findings were correlated with clinical outcomes with an average of a 49-month follow-up. FOXO1 break-apart by FISH was positive in 4 of 6 (66%) ARMS and 2 of 5 (40%) RMS-NOS cases. All other cases, including all ERMS, were negative. RT-PCR assay confirmed all FISH results. While 2 of 6 (33%) RMS patients with a FOXO1 break-apart died of the disease, there were no deaths among the patients with negative result. The FOXO1 gene break-apart FISH probe is a simple and accurate tool to detect the translocations associated with ARMS. As characteristic genetic alterations of ARMS can be identified in 40% of RMS-NOS cases in our study, the FISH assay would provide an additional useful tool in the diagnosis and prognosis of ARMS, and an alternative to RT-PCR.