To reveal insights into the inhibition of BCR-ABL and its mutants, structure-based computing methods, such as docking, molecular dynamics (MD) simulation, the molecular mechanics generalized born surface area (MMGBSA), and biological characterizations, were employed to analyze two main pharmacophore zones and two related regions of imatinib derivatives. The hydrophobic and halogen interactions formed by the trifluoromethyl, as well as T-shaped π-π interactions formed by the pyrimidine, were confirmed. For the imatinib derivatives, the impacts of the amide moiety (region A) and the pyridine (region B) on the formed interactions were explored. To reveal insights into the inhibition of BCR-ABL mutants, the bioactivities of imatinib, nilotinib and flumatinib against BCR-ABL mutants were evaluated, and a point mutant (Y253F) of BCR-ABL was simulated. The results of our structure-based analysis and biological characterization of imatinib derivatives towards the inhibition of wild-type BCR-ABL and its mutants may provide new ideas for the design of imatinib analogs with potent activity.