The detection of hyperdiploidy (clones with >46 chromosomes) in the bone marrow of patients with acute lymphoblastic leukaemia (ALL) is important because of the prognostic impact of this finding. The high hyperdiploid (HeH) subgroup with 51-68 chromosomes is associated with the best outcome, followed by the low hyperdiploid (HeL) subgroup with 47-50 chromosomes and the triploid/tetraploid (TT) subgroup with >68 chromosomes, which do less well. We present a strategy for the use of fluorescence in situ hybridization (FISH) with chromosome-specific probes to detect hyperdiploidy in interphase cells and to assign cases to a ploidy subgroup. By using a model population of 252 cases, it was seen that ten chromosomes (X, 4, 6, 8, 10, 14, 16, 18, 20, and 21) used in particular combinations and applied in a step-wise manner enabled the detection of 94% of hyperdiploid cases and gave an accurate prediction of ploidy subgroup in 96% of these cases. The detection and classification of each case required the use of four to six probes over two or three steps. Confirmation that this strategy will achieve this level of detection in other hyperdiploid populations was demonstrated by using 250 published karyotypes. This strategy has an application in detecting missing or hidden hyperdiploid cases among cases with failed or normal cytogenetics.