Imatinib mesylate is currently the drug of choice for all phases of chronic myeloid leukemia (CML). Despite the initial high rates of hematological and cytogenetic responses, many patients develop resistance. We prospectively studied 40 patients with CML with primary cytogenetic resistance to imatinib mesylate. A semi-nested reverse transcriptase-PCR approach was used for amplification of the ABL kinase domain, which was then subjected to direct sequencing for the detection of point mutations. Expression of BCR-ABL transcripts was quantified using the real-time Taqman assay, and BCR-ABL gene amplification was detected using fluorescence in situ hybridization. Twelve different point mutations were detected in 18/40 (45%) patients. Five patients had mutations in the P-loop region and 13 had mutations in other regions of the BCR-ABL kinase domain. Progression-free survival at 2 years was inferior for patients with mutations compared to those without mutations (72% vs. 95%, p < 0.0045). The BCR-ABL fusion gene was over-expressed in five patients (5/18); the mean BCR-ABL/ABL ratio was 75.38 vs. 28.72 for imatinib responders, p < 0.001. Amplification of the BCR-ABL fusion gene was detected in 4/40 (10%) patients. Point mutation was the major mechanism of primary cytogenetic resistance to imatinib mesylate in the present study. Patients with mutations had inferior progression-free survival compared to those without mutations. Regular monitoring for the presence of point mutations in the ABL kinase region may identify such patients early, with an opportunity to intervene.