Synovial sarcomas are highly malignant tumors of soft tissue which are characterized by the t(X;18) resulting in SYT-SSX fusion transcript production. Diagnosis of these tumors based on histology can be challenging, particularly when minimal biopsy specimens are presented to the pathologist. Demonstration by molecular methods of SYT-SSX transcripts is a useful adjunct for diagnosis in these situations. We have developed an assay, which combines one-step RT-multiplex PCR with capillary electrophoresis to detect and genotype the SYT-SSX transcripts from synovial sarcomas. Small amplicons from chimeric transcripts as well as GAPD transcripts are differentially labeled with fluorophores, allowing detection and size discrimination by capillary electrophoresis. In a study of 32 formalin-fixed soft tissue tumor specimens, the assay detected chimeric transcripts from 17/22 (77%) synovial sarcomas. All five assay negative specimens yielded no intact RNA as evidenced by lack of a GAPD amplicon. Chimeric transcripts were not detected in 9/9 malignant peripheral nerve sheath tumors or 1/1 epithelioid sarcoma. Representative amplicons were sequenced and confirmed the genotype results obtained by capillary electrophoresis. One-step RT-multiplex PCR combined with capillary electrophoresis is a rapid and accurate method for the detection and genotypic classification of SYT-SSX transcripts from fixed tissue specimens.