T-cell prolymphocytic leukemia (T-PLL) is a rare malignant proliferation of lymphoid cells with a postthymic phenotype. Previous cytogenetic and molecular studies reported complex karyotypes with recurrent chromosomal abnormalities, including translocations involving either TCL1 at 14q32.1 or MTCP1 at Xq28, inactivation of the ATM gene by deletion and/or mutation, and isochromosomes 8. For extensive study of chromosomal imbalances in T-PLL, we analyzed 22 tumoral DNAs using comparative genomic hybridization (CGH). Abnormal CGH profiles were detected in all cases, demonstrating highly recurrent gains and losses and largely extending the abnormalities previously established. Only a few nonrecurrent abnormalities were observed, in contrast to the genetic instability anticipated from ATM inactivation. Nine recurrent regions of loss were identified at 8p (frequency 86%), 11q (68%), 22q11 (45%), 13q (41%), 6q (36%), 9p (27%), 12p (23%), 11p11–p14 (23%), and 17p (23%), as well as four regions of gain at 8q (82%), 14q32 (50%), 22q21–qter (41%), and 6p (23%). Several recurrent chromosomal abnormalities were simultaneously present in each case (mean, 5.7; up to 10), none being mutually exclusive of another. Fluorescence in situ hybridization analysis confirmed and extended 22q11 and 13q losses, giving final frequencies of 55% and 45%, respectively. Analysis of one case over a 7-year period confirmed the overall genetic stability of T-PLL and showed that tumor progression was associated with the onset of a few chromosomal abnormalities. This study establishes a complex pattern of highly recurrent chromosomal abnormalities in T-PLL, including some, such as chromosome 13 deletion, commonly found in other lymphoid malignancies. © 2001 Wiley-Liss, Inc.