Translocations can be detected using fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tissues. Recently, a commercially available FKHR (13q14) dual-color, break-apart rearrangement probe has been developed. However, the advantages of using this probe have not been reported. This study demonstrated the usefulness of this probe for the clinical diagnosis of rhabdomyosarcomas (RMS). We studied 33 RMS (19 embryonal rhabdomyosarcomas [ERMS], including three sclerosing-type RMS, and 14 alveloar rhabdomyosarcomas [ARMS]). Fluorescence signals were detected for 18 of the 19 (94.7%) ERMS and 13 of the 14 (92.8%) ARMS. A split-signal pattern was detected in 12 of 13 (92.3%) ARMS but was not detected in any of the ERMS, including the three sclerosing-type RMS. Amplification and polyploidy were present in both the ERMS and the ARMS. Our FISH study highlighted the excellent performance of the presently reported commercial break-apart probe for the detection of FKHR gene rearrangements in RMS. Because amplification and polyploidy were detected in both the ERMS and the ARMS, sufficient care should be taken when counting the nuclear signals. No rearrangements of the FKHR gene were found in any of the three sclerosing-type RMS when examined using a FISH assay, supporting the hypothesis that sclerosing RMS can be included as an ERMS.