Mutations in the kinase domain of the abl gene are believed to be responsible for resistance to imatinib in patients with chronic myeloid leukemia (CML). These mutations are thought to impair binding of imatinib to the ATP-binding domain of the Abl protein. Most studies test for these mutations using peripheral blood cells, because of the assumption that resistant leukemic cells in peripheral blood must contain representative mutational changes. Frequently, however, progression of the disease manifests with increased blasts in bone marrow (BM) and not in peripheral blood (PB). We investigated the potential of detecting mutations in BM rather than PB cells in patients with CML. Because we previously reported that PB plasma is enriched with tumor-specific circulating protein, DNA, and RNA, we also compared PB plasma to BM and PB cells for detecting mutations in the Bcr-Abl kinase domain. Analysis was conducted with reverse-transcription PCR targeting an 863-bp region of the bcr-abl allele, followed by sequencing. Simultaneous analysis of plasma, PB cells, and BM cells from 41 CML patients with clinical resistance to imatinib detected a total of 28 distinct mutations, 3 of which are novel mutations. The 3 sample types showed 80% concordance in detecting mutations (33 of 41), and all patients in this group had at least 1 mutation detected in plasma and BM cells. Only 2 of the patients with concordant results harbored multiple mutations (G250E and F359I in both cases). Among the 8 samples with discordant results, 13 different mutations were detected in plasma, 11 in BM cells, and 9 in PB cells. Two of the 8 patients with discordant results showed a mutation in BM and plasma but not in PB cells (wild-type). In 5 of the remaining 6 patients with discordant results, additional mutations not found in PB were detected in plasma. In 1 patient, the T315I mutation was detected in plasma and BM but not PB cells. Interestingly, 2 patients had mutations detected in a small population of PB cells but not in BM cells or plasma, in addition to more dominant mutations detected in plasma and BM cells. We detected no evidence of mutation in the plasma of 45 previously untreated patients with CML. These results confirm the presence of multiple clones in patients with CML and resistance to imatinib and suggest that plasma might be an adequate alternative to bone marrow for detecting mutations in the kinase domain of the Bcr-Abl fusion gene.