The search for chromosomal translocations in de novo cases of childhood acute lymphoblastic leukaemia (ALL) is crucial for the selection of the appropriate therapeutic protocol. In this work, we describe a new method - one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) - to screen for prognostic significant translocations in childhood ALL. Our approach involves a single PCR reaction for the simultaneous detection of the molecular rearrangements resulting from the t(9;22), t(12;21), t(4;11) and t(1;19), with a turnaround time of less than 24 h. This assay proved to be highly sensitive, specific, reproducible and easy to implement in a routine genetics laboratory.