The most common treatment of chronic lymphocytic leukemia (CLL) is the alkylating agent chlorambucil (CLB), with or without prednisone. In the present study, our aim was to evaluate whether treatment with CLB for more than one year induced genetic changes manifested by comparative genomic hybridization (CGH) as new chromosomal aberrations. We also studied whether CLB affected the pattern of replication by using fluorescence in situ hybridization (FISH). We found a similar rate of asynchronous pattern of replication in both treated and untreated patients with CLL. Most of the aberrations found with CGH were previously reported in CLL. More prognostically unfavorable aberrations and more cases with genetic changes were found in the treated group. The changes found were not typical of the secondary genetic aberrations associated with alkylating agents. Thus, we conclude that treatment of CLL with CLB for at least a year does not affect the parameters analyzed in this study. Longer studies are needed to further explore the effects of alkylating agents on normal and malignant cells.