The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)J-mediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usage-defined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers.