Alveolar rhabdomyosarcoma (ARMS) is an aggressive neoplasm with unique t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations, resulting in PAX3/FOXO1 and PAX7/FOXO1 fusion genes, in approximately 80% of cases. These translocations and their gene fusions have not been identified in other neoplasms, making their identification an attractive target for applying ancillary diagnostic techniques such as fluorescence in situ hybridization (FISH). We report our experience with a dual-color break-apart FISH probe for the detection of FOXO1 (13q14) rearrangements in neoplasms within the differential diagnosis of ARMS, using routinely processed formalin-fixed, paraffin-embedded tissues. A total of 52 sarcomas were analyzed including ARMS (n = 25), embryonal rhabdomyosarcomas (n = 8), neuroblastoma (n = 1), desmoplastic small round cell tumors (n = 2), Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET; n = 15), and round cell liposarcoma (n = 1). Cytogenetics and/or reverse transcription polymerase chain reaction data were available on a subset of the ARMS (n = 11) and EWS/PNET cases (n = 5). A minimum of 100 interphase tumor nuclei were evaluated for the presence of intact or translocated signals. FOXO1 gene rearrangements were identified in 88% (22/25) of ARMS (mean: 91% positive cells/case; range: 50% to 100%), whereas no rearrangements were detected in the other neoplasms examined (mean: 1.4% positive cells/case; range: 0% to 4%). FOXO1 (13q14) FISH on formalin-fixed, paraffin-embedded tissues samples showed excellent concordance with reverse transcription polymerase chain reaction and cytogenetic analyses in ARMS cases, demonstrated excellent specificity (100%) when applied to potential mimickers such as EWS/PNET, and played an important role in the differential diagnosis of small round cell tumors.