Background: Chronic Lymphocytic Leukemia (CLL) is a common hematologic malignancy characterized by the proliferation of mature B lymphocytes. The disease accounts for a significant proportion of adult leukemias, with an estimated annual incidence of 4.5 cases per 100,000 individuals. Despite its prevalence, CLL can present with diverse clinical manifestations, posing diagnostic and therapeutic challenges. One of them is clonal evolution, which refers to the acquisition of genetic mutations by leukemic B cells over time, resulting in the development of subclones with distinct genetic characteristics. As the disease progresses, different sub-clones may become dominant, contributing to disease relapse and resistance to treatment. Methods: This case report presents the clinical evolution of a 65-year-old patient diagnosed with CLL, focusing on clonal evolution and treatment response. The patient was first diagnosed with CLL in 2017, with flow cytometry revealing CLL characterized by negative CD34 and positive CD49d markers. Fluorescence in situ hybridization (FISH) analysis revealed trisomy 12 in 65% of cells. The patient received 5 cycles of chemotherapy out of 6 prescribed cycles. The therapy was withheld due to thrombocytopenia. Although the patient received his last chemotherapy in March 2018, he continued to exhibit persistent pancytopenia during blood work conducted in July 2023, prompting a bone marrow biopsy and FISH analysis. Results demonstrated a decrease to 57% in trisomy 12 prevalence, accompanied by the emergence of a NOTCH1 mutation. In flow cytometry, CD49d was negative, indicating a change in the phenotype of the leukemic cells as well. Results: The patient's CLL exhibited dynamic clonal evolution, as evidenced by changes in cytogenetic abnormalities and the acquisition of a NOTCH1 mutation over the course of the disease. Despite initial responses to treatment, including a complete remission with incomplete bone marrow recovery after chemoimmunotherapy, the disease eventually progressed, necessitating the initiation of targeted therapy. Conclusion: This case highlights the dynamic nature of CLL and the impact of clonal evolution on disease progression and treatment response. Molecular monitoring, including FISH analysis and mutation profiling, is essential for identifying changes in disease biology and tailoring treatment strategies accordingly. Understanding the underlying mechanisms of clonal evolution in CLL is crucial for optimizing treatment outcomes and improving patient care.