The identification of specific chromosome abnormalities in acute myeloid leukemia (AML) is important for the stratification of patients into the appropriate treatment protocols. However, a significant proportion of diagnostic bone marrow karyotypes in AML is reported as normal by conventional cytogenetic analysis and it is suspected that these karyotypes may conceal the presence of diagnostically significant chromosome rearrangements. To address this question, we have developed a novel 12-color fluorescence in situ hybridization (FISH) assay for telomeric rearrangements (termed M-TEL), which uses an optimized set of chromosome-specific subtelomeric probes. We report here the application of the M-TEL assay to 69 AML cases with apparently normal karyotypes or an isolated trisomy. Of the 69 cases examined, 3 abnormalities were identified, all in the normal karyotype group. The first was a t(11;19)(q23;p13), identified in an infant with AML-M4. In 2 other young patients with AML (< 19 years), an apparently identical t(5;11)(q35;p15.5) was identified. Breakpoint mapping by FISH and reverse transcriptase polymerase chain reaction (RT-PCR) analysis confirmed that this was the same t(5;11) as previously identified in 3 children with AML, associated with del(5q) and resulting in the NUP98-NSD1 gene fusion. The t(5;11) was not detected by 24-color karyotyping using multiplex FISH (M-FISH), emphasizing the value of screening with subtelomeric probes for subtle translocations. This is the first report of the t(5;11)(q35;p15.5) in association with an apparently normal karyotype, and highlights this as a new, potentially clinically significant chromosome rearrangement in childhood AML.