Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that 1 mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.