Purpose: The purpose of this study was to assess the biological significance of lactate dehydrogenase (LDH), T315I mutation and treatment options in newly diagnosed and relapsed patients with chronic myeloid leukemia (CML). Methods: Our clinical-analytical and descriptive study enrolled 27 patients with different phases of CML, who were followed up and treated at the Institute of Oncology between 1995-2020. Venous blood samples were taken for LDH measurement, molecular screening and detection of T315I mutation of the ABL gene in order to investigate the biological significance of the increased LDH values and T315I mutation. CML patients underwent chemotherapy with alkylating agents, antimetabolites and tyrosine kinase inhibitors (TKIs). Results: The patient age ranged from 20 to 67 years (mean 51.3±2,14). The diagnosis of CML was established in the late chronic phase in 25 (92.6%) patients. The quantitative real-time PCR revealed p210 transcript of the BCR-ABL chimeric gene in all cases, with the range of 23.17-100% and median value of 74.73±3.21%. LDH at diagnosis ranged between 169-1609.4 U/L and was increased in 14 (63.6%) patients, especially in those with leukocytosis over 100×109/l. The complete cytogenetic and complete or major molecular responses were recorded under treatment with different generations of TKIs in 16 (59.3%) cases, including 3 cases with T315I mutation. Relapses occurred in 10 (71.4%) patients with initially increased LDH values and in 5 of 6 patients with T315I mutation. One (3.7%) patient with T315I mutation evolved into the acute phase disease, and achieved the complete hematological response after treatment with ponatinib, a 3rd generation TKI. The survival of patients from the disease onset till the last monitoring visit ranged between 21 and 234.8 months (median 93.97±4.52). Conclusions: The increased LDH values may indicate the activity at diagnosis and relapse of CML. In our study T315I mutation of the ABL gene and the increased values of LDH were associated with a higher rate of relapses and resistance to imatinib. Notwithstanding the treatment line and in relapses TKIs improve considerably the survival and ECOG-WHO score of CML patients.