Background: EGFR activating mutations predict sensitivity to tyrosine kinase inhibitors (TKIs) in NSCLC. Although initial responses are commonly observed, patients inevitably progress as a consequence of acquired resistance. Secondary mutations in the EGFR domains (Gainor JF, Shaw AT. J Clin Oncol 2013;31:3987) or in cross-talking pathways (i.e., KRAS, Martin P, et al. J Thorac Oncol 2013;8:530) have been detected in biopsies of resistant cancers. However, the location of the tumor and the risk of complications are serious limitations to re-biopsies in NSCLC. Alternatively, detection of somatic mutations in circulating cell-free DNA (ccfDNA) in plasma could be instrumental to a better understanding of the dynamics of the genetic shift of tumors under stress conditions caused by drug treatments. Methods: Twenty-eight NSCLC patients displayed an activating EGFR mutation or ALK translocation in the primary tumor and received a TKI against EGFR (gefitinib or erlotinib, n= 25) or ALK (crizotinib, n= 3). Blood (5 ml) was collected after tumor progression and DNA was extracted from plasma using QIAamp circulating nucleic acid kit and tested for EGFR and KRAS mutations using Rotor-Gene Q PCR (Qiagen, Valencia, CA) or digital droplet PCR (Bio-Rad, Hercules, CA). Results: EGFR mutations of the primary tumor were confirmed in ccfDNA of 35.7% patients after progression under TKIs (L858R 7.1%, exon 19 insertions/deletions 28.6%). The EGFR T790M mutation was in 10.7% of patients’ ccfDNA. Interestingly, 10 (35.7%) patients displayed a codon 12 KRAS mutation in ccfDNA, including all patients with ALK+ tumors, after TKI treatment. The KRAS mutations were not detectable in their primary tumor using conventional diagnostic approaches. Pre-treatment plasma samples were available in only two patients and KRAS mutations were not detected. Conclusions: The observation of KRAS mutations in plasma of patients with tumors carrying EGFR mutations or ALK rearrangements after TKI treatment suggests an important role of this oncogene in acquired resistance.