Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of two copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.