Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is effective in EGFR T790M positive non-small-cell lung cancer (NSCLC). Despite the efficacy of osimertinib, patients inevitably develop resistance and the mechanisms of osimertinib resistance are heterogeneous. Here, we report that a lung adenocarcinoma patient with EGFR L858R mutation who was treated with second-line osimertinib therapy acquired multiple resistance to osimertinib by the non-invasive circulating tumor DNA (ctDNA) genotyping. This case provides the possible mechanisms of osimertinib resistance that occur during the disease progression and supports the longitudinal monitoring of ctDNA for the detection of novel acquired resistance and tumor heterogeneity.