Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) type is a distinct clinicopathological disease entity in the category of extranodal marginal zone B-cell lymphoma. Recently, we and others have shown that the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18 are fused as a result of t(11;18)(q21;q21) in MALT lymphomas. Here we report a detection assay that can be used for formalin-fixed, paraffin-embedded specimens. It consists of a multiplex one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) followed by three parallel multiplex nested polymerase chain reactions. Eight variants of the fusion transcripts have been reported to date. When these variants were used as positive controls, all were successfully detected. The subsequent direct sequencing confirmed the results. Using this rapid and simple method, we could detect API2-MALT1 fusion transcripts in 5 of 15 (33%) archival cases of MALT lymphoma for a frequency comparable with those of RT-PCR assays using frozen materials. The lung was the preferential anatomical site of origin of MALT lymphomas harboring API2-MALT1 fusion. No fusion transcript was detected in any of 20 high-grade B-cell lymphomas. Our multiplex RT-PCR assay, which can be used for routinely-processed paraffin samples, should serve as a useful molecular tool for clarifying the clinicopathological significance of API2-MALT1 fusion in MALT lymphoma.