Chromosomal rearrangements in childhood acute lymphoblastic leukemia (ALL) play an important role in the identification of clinical relevant subgroups. For rapid and easy detection of the clinically most important gene rearrangements, a nested multiplex reverse transcriptase polymerase chain reaction (multiplex PCR) was developed. This multiplex PCR enables the detection of M-BCR/ABL, m-BCR/ABL, TEL/ AML1, and MLL/AF4 fusion transcripts in one PCR reaction. However, the existence of splicing variants and different breakpoints on the DNA level hampers the discrimination of the rearrangements by their fragment size on an agarose gel. Therefore, one of the internal primers of each translocation (ABL-2, TEL-2, AF4-2) was labeled with a characteristic fluorescent dye, and an automatic fluorescence-based DNA fragment analysis was performed. The sensitivity of this multiplex PCR is in the same range as that of the corresponding single PCR reaction and allows a fast screening for the detection of therapy-relevant rearrangements, with a high turnover of samples.