Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xeno-patient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacological analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next generation TRKA inhibitors.