Kirsten rat sarcoma viral oncogene homolog (KRAS) is a small GTPase protein which plays an important role in the treatment of KRAS mutant cancers. The FDA-approved AMG510 and MRTX849 (phase III clinical trials) are two potent KRASG12C-selective inhibitors that target KRAS G12C. However, the drug resistance caused by the second-site mutation in KRAS has emerged, and the mechanisms of drug resistance at atom level are still unclear. To clarify the mechanisms of drug resistance, we conducted long time molecular dynamics simulations (75 μs in total) to study the structural and energetic features of KRAS G12C and its four drug resistant variants to inhibitors. The combined binding free energy calculation and protein−ligand interaction fingerprint revealed that these second-site mutations indeed caused KRAS to produce different degrees of resistance to AMG510 and MRTX849. Furthermore, Markov State Models and 2D-free energy landscapes analysis revealed the difference in conformational changes of mutated KRAS bound with and without inhibitors. Furthermore, the comparative analysis of these systems showed that there were differences in their allosteric signal pathways. These findings provide the molecular mechanism of drug resistance, which helps to guide novel KRAS G12C inhibitor design to overcome drug resistance.