Acquired imatinib resistance in advanced Philadelphia-positive acute lymphoblastic leukemia (Ph(+) ALL) has been associated with mutations in the kinase domain (KD) of BCR-ABL. We examined the prevalence of KD mutations in newly diagnosed and imatinib-naive Ph(+) ALL patients and assessed their clinical relevance in the setting of uniform frontline therapy with imatinib in combination with chemotherapy. Patients enrolled in the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) trial ADE10 for newly diagnosed elderly Ph(+) ALL were retrospectively examined for the presence of BCR-ABL KD mutations by denaturing high-performance liquid chromatography (D-HPLC), cDNA sequencing, and allele-specific polymerase chain reaction (PCR). A KD mutation was detected in a minor subpopulation of leukemic cells in 40% of newly diagnosed and imatinib-naive patients. At relapse, the dominant cell clone harbored an identical mutation in 90% of cases, the overall prevalence of mutations at relapse was 80%. P-loop mutations predominated and were not associated with an inferior hematologic or molecular remission rate or shorter remission duration compared with unmutated BCR-ABL. BCR-ABL mutations conferring high-level imatinib resistance are present in a substantial proportion of patients with de novo Ph(+) ALL and eventually give rise to relapse. This provides a rationale for the frontline use of kinase inhibitors active against these BCR-ABL mutants.