In chronic lymphocytic leukemia (CLL), the acquisition of new genomic aberrations during the disease course (clonal evolution) is thought to be an infrequent phenomenon but comprehensive analyses are limited. Genomic aberrations were analyzed by fluorescence in situ hybridization (FISH) at various time points during the disease course of 64 CLL patients. Results were correlated with the mutation status of the immunoglobulin heavy-chain variableregion genes (VH) and clinical characteristics. Following a median observation time of 42.3 months (range 23.2–73) after first genetic study, 11 out of the 64 (17%) patients showed clonal evolution with the following newly acquired aberrations: del (17p13)(n= 4), del (6q21)(n= 3), del (11q23)(n= 2),+(8q24)(n= 1), and evolution from monoallelic to biallelic del (13q14)(n= 3). Interestingly, clonal evolution only occurred among cases with unmutated VH status. The group with clonal evolution showed a higher rate of progression in Binet stage (82% vs. 28%), a possibly greater need for treatment (91% vs. 62% previously untreated patients received their first therapy), and a higher hazard rosk of death (HR= 2.97, 95% CI 1.40–6.27, p= 0.004) in multivariable analysis. The estimated median survival time after the occurrence of clonal evolution was 21.7 months. Expansion of the clone with del (17p13) was observed in all patients during treatment, indicating in vivo resistance to therapy. In multivariable Andersen-Gill regression analysis, clonal evolution was identified as an independent prognostic factor for overall survival. Clonal evolution only occurred in CLL with unmutated VH indicating to karyotypic instability as a pathomechanism. Acquisition of genomic aberrations was associated with poor outcome based on multivariable analysis. In vivo resistance to chemotherapy of CLL clones with del (17p13) emphasizes the need for alternative treatment approaches in these patients.