Resistance to imatinib in CML may occur through mutations of the BCR-ABL kinase domain. We investigated the occurrence of mutations in 159 pts treated with imatinib by direct sequencing. Pts had been treated with imatinib for a median of 32 months (mo) (range, 4 to 60 mo). 52 mutations were identified in 49 (31%) pts: 3 pts had 2 different mutations; in 2 of them they appeared simultaneously. Mutations mapped to 23 different amino acid residues, with the most frequent locating to the P-loop (n=19; 39%), and the residues in imatinib binding pocket (n=12; 25%). The single most common mutations were G250E (n=8) and F317L (n=7). At the time imatinib was started, 34 pts (70%) were in chronic phase (CP) (13 previously untreated, 21 post-IFN failure), 14 (28%) in accelerated phase (AP), and 1 (2%) in lymphoid blast phase (BP). Three pts (2 AP, 1BP) had clonal evolution. Best response to imatinib for pts in CP was: cytogenetic (CG) response complete (CGCR) in 13 (38%), partial (CGPR) in 8 (24%), minor in 1 (3%), and hematologic response only in 12 (35%) (11 complete, 1 partial). For pts in AP, best response was: CGCR in 5 (36%), CGPR in 3 (21%), minor in 1 (7%), and hematologic only in 5 (36%). The pt in BP had achieved CGCR. At the time mutations occurred, 47 (96%) pts had lost their hematologic or cytogenetic response, and clonal evolution had occurred in 15 (68%) pts. Among pts who were initially treated in CP, 9 had transformed to AP and 3 to BP. Two pts treated in AP had progressed to BP. In 2 (4%) pts (1 CP, 1 AP) the mutation (F317L, F311I) was identified while in CGCR remission; both pts have remained in CG CR 2 and 4 mo, respectively after the mutations was identified. Five patients died (4 AP, 1 CP) after a median of 7 mo (1–20 mo) following the detection of mutations. Their mutations included F317L (n=2), M244V, F359V, H396R (1 each). Thus, after a median follow-up of 6 mo (range, 1–20 mo) from detection, only 1 of 19 pts with P-loop mutations has died versus 4 of 30 with non-P-loop mutations. The estimated 15-months survival rates were 95% and 83%, respectively (p=0.50). We conclude that 1) mutations occur frequently among pts with acquired resistance to imatinib; 2) in few instances they can be identified in pts in CGCR; and 3) P-loop mutations may not be associated with worse outcome compared to other mutations.