Myxoid liposarcoma with or without a round cell component is the most common subtype of liposarcoma. The diagnosis of myxoid liposarcoma could be challenging with histology, as a variety of soft tissue tumors with myxoid change might mimic myxoid liposarcoma, especially on small biopsy tissues. Chromosomal translocations of t(12,16) (q13;p11) and t(12;22) (q13;q12), rendering gene fusions of DDIT3 (previously CHOP) with FUS and EWSR1, have been found to be characteristic of myxoid liposarcoma, and were identifiable in more than 95% cases. These genetic alterations, therefore, are ideal as molecular markers to facilitate the diagnosis of this type of tumor. DDIT3 (12q13) dual-color break-apart rearrangement probe for fluorescence in situ hybridization has been commercially available. However, its consistency with DDIT3-associated gene fusion and its clinical use, including sensitivity and specificity, have not been adequately evaluated. In this study, we assessed the locus specificity of the probe on metaphase, and then tested it on 8 cases of myxoid liposarcoma, 12 cases of other sarcomas, and 18 cases of tumors with myxoid differentiation. All 8 myxoid liposarcomas showed DDIT3 gene break-apart, whereas all 12 other sarcomas were negative. All the cases with DDIT3 break-apart also showed FUS-DDIT3 fusion by reverse transcription-polymerase chain reaction, with 100% consistency. In addition, the FISH assay has been clinically applied on 18 myxoid tumors with promising outcome. In conclusion, FISH with DDIT3 break-apart probe is a highly sensitive and specific assay for detection of DDIT3-associated gene fusions, and therefore is a valuable adjunct in diagnosis or differential diagnosis of myxoid liposarcoma.