Oligoclonal B cell proliferation, as defined by the presence of more than one leukemic clone, has been detected in approximately 20% to 30% of patients with acute lymphoblastic leukemia (ALL) using PCR or Southern blotting. An accurate assessment of these populations is required to avoid false negative measurements of minimal residual disease (MRD) in follow-up bone marrow (BM) samples of ALL patients. In this study, we analysed 29 ALL patients with two or more immunoglobulin heavy (IGH) chain gene rearrangements in the presentation samples using IGH fingerprinting PCR and sequence analysis. Thirty-nine (51%) of 76 sequences (from 15 patients), shared no VNDNJ homology (ie different CDR3 regions). In the remaining 14 patients, at least two related VH sequences were identified in each patient (identical DNJ sequences). Numerical abnormalities of chromosome 14 was detected in 10 patients. Eight patients were analysed at presentation and relapse. In four of them, expansion of a minor presentation-clone was detected at relapse while the major presentation clone disappeared, confirming 'subclonal evolution'. Finally, in our cohort of patients, the presence of related or unrelated IGH clones did not influence overall survival.