Background: Osimertinib (Osi) is the key standard therapy for patients (pts) with chemo-naive advanced non-small cell lung cancer (NSCLC) harboring sensitizing epidermal growth factor receptor (EGFR) mutations. However, many of cases treated with Osi experience the disease progression (PD). Therefore, understanding first-line osimertinib resistance mechanisms is essential for future therapeutic strategies. Here, we addressed the underlying mechanisms of resistance to Osi using paired plasma DNA samples and particularly focused on early PD (PD within 6 months) cases. Methods: This trial is the multicenter prospective research, and we prospectively collected plasma DNA samples prior Osi treatment and at PD. Next-generation sequencing (NGS) implemented with the molecular barcoding method was performed to analyze gene mutations in 197 cancer-associated genes. Then using these ct-DNA, we analyzed the Osi resistance by ultra-sensitive NGS. In this interim analysis, we specially analyze the early PD (PD within 6 months) cases. Results: One hundred eighty-eight pts were enrolled between May 2019 and January 2021. Among these pts, 125 (66%) pts were female, 96 (51%) were EGFR exon 19 deletion mutation, and 109 (58%) were never smoker. Seventy-eight pts experienced PD and 36 pts experienced early PD at interim analysis. Baseline shedders, patients with plasma positivity for EGFR-activating mutation were more frequently observed in early PD pts than non-PD (86.1% vs 63.4%, p = 0.009). Early PD patients had significantly more co-occurring gene mutations along with EGFR mutations than non-early PD patients (2.89±1.49 vs 1.97±1.37, p = 0.002). Most notably, 3 among 36 early PD pts and 8 among 78 PD pts had significant reduction of plasma EGFR mutant allele frequency at PD than that at baseline. Among those, 1 had germline BRCA1 mutation and 1 had BRINP3 mutation in 3 early PD pts, and 1 had germline BRCA2 mutation, and 1 had germline TP53 mutation in 5 PD pts excluding early PD pts. Co-occurring TP53 mutations were not relevant to early PD in this study cohort. Conclusions: Germline mutations in BRCA1/2, TP53 and BRINP3 may confer early and unprecedented resistance to Osi monotherapy. Plasma-based serial comprehensive gene profiling may help predict and identify patients unlikely to benefit from Osi treatment, though further study is warranted. Clinical trial information: jRCTs031180051.