Dasatinib and nilotinib are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib-resistant chronic myeloid leukemia (CML) clones with BCR-ABL kinase domain (KD) mutations, except T315I. We assessed for changes in the BCR-ABL KD mutation status in 112 patients with persistent CML who received a second-generation TKI after imatinib failure. Sixty-seven different KD mutations were detected before the start of therapy with a second TKI, with T315I seen in 15%. Equal numbers of patients received nilotinib or dasatinib following imatinib, and 18 received 3 TKIs. Response rates were similar for patients with and without mutations, regardless of mutation site except for T315I. Overall, 29 patients (26%) developed new KD mutations after therapy with a second (n = 24) or third (n = 5) TKI, but only 4 (4%) developed T315I. In 73% of cases, the KD mutations that persisted or developed following switch to new TKI were at sites also found in prior in vitro TKI mutagenesis assays. Although there is only a mild increase in mutation frequency with sequential TKI treatment, novel mutations do occur and mutation regression/acquisition/persistence generally reflects the in vitro differential sensitivity predicted for each TKI. In this study, there was no marked increase in development of T315I.