The t(6;9)(p23;q34)-DEK/CAN fusion occurs with an incidence of 1-5% in adult patients with acute myelogenous leukemia (AML) and tends to have an unfavorable prognosis at diagnosis. Due to the subtle appearance of this chromosome rearrangement, both initial detection and minimal residual disease (MRD) tracking by conventional karyotyping can be difficult. Unfortunately, no commercial or previously published fluorescence in situ hybridization (FISH) strategies exist for this recurrent anomaly. We have developed a highly sensitive assay using dual-color, double-fusion FISH (D-FISH), which can be used both for initial detection and MRD monitoring. We analyzed archived bone marrow samples from 15 patients with a previously identified t(6;9)(p23;q34) and 10 corresponding post-treatment samples. The results demonstrate that our D-FISH method effectively identified all abnormal samples, including a low-level MRD sample that was considered to be normal by conventional cytogenetic analysis. Normal value ranges were established from 30 negative controls to be < 0.6% when 500 interphase nuclei were analyzed. The development of this sensitive D-FISH strategy for the detection of the t(6;9)(p23;q34) adds to the AML FISH testing repertoire, and is effective in the detection of low-level disease in post-treatment samples in these patients.