Summary Introduction This study investigates the benefits of using multiplex reverse transcriptase‐PCR (RT‐PCR) in addition to standard karyotyping during the initial evaluation of acute leukemia. Methods A total of 1114 consecutive specimens from patients with acute leukemia were tested using a commercial multiplex RT‐PCR kit (HemaVision, DNA Diagnostic). NPM1 and CEBPA mutations were selectively tested in acute myeloid leukemia (AML) patients with multiplex RT‐PCR negativity. Results In specimens with optimal cytogenetics, the frequency of recurrent translocations was 31.3%, and cryptic translocations were detected in 2.1% of samples. The concordance rate between karyotyping and multiplex RT‐PCR was 97.5%. In addition to the established functions, we demonstrated the additional benefits of multiplex RT‐PCR, including successful molecular characterization, even in cytogenetically suboptimal specimens (5.7%); detection of submicroscopic aberrations (1.0%); detection of rare but potentially significant translocations or variants (2.5%); selection of AML candidates for mutation analysis (68.3%); and finally exclusion of recurrent translocations in patients with acute lymphoblastic leukemia or mixed phenotype acute leukemia (22.5%). Conclusion We reconfirmed the accuracy and reliability of multiplex RT‐PCR for diagnosing acute leukemia and demonstrated additional advantages of this system for the initial evaluation of acute leukemia. Thus, multiplex RT‐PCR is worth considering in diagnostic testing of acute leukemias.