AbstractEpidermal growth factor receptor (EGFR) exon 20 insertion mutations (Exon20ins) account for 4–12% of all EGFR mutations in non-small cell lung cancer (NSCLC) patients. Data on the differences in clinical characteristics between patients with Exon20ins and major mutations (M-mut) such as exon 19 deletion and L858R are limited. We retrospectively reviewed advanced NSCLC patients with EGFR mutations, who were treated with systemic therapy between January 2011 and December 2019. We identified 23 patients with Exon20ins and 534 patients with M-mut. In Exon20ins patients, the median age was 60 (range 27–88) years, and females and never-smokers were predominant. Clinical characteristics were similar in the two groups. In Exon20ins patients, 17 patients received platinum doublet as first-line therapy, and the overall response rate (ORR) and median progression-free survival (mPFS) were 11.8% and 8.9 months. Additionally, seven patients received conventional EGFR-tyrosine kinase inhibitors (TKIs), and eight patients anti-PD-1 antibodies in any-line therapy. ORR and mPFS of EGFR-TKIs and anti-PD-1 antibodies were 0%, 2.2 months and 25%, 3.1 months, respectively. Overall survival was significantly shorter in Exon20ins patients than in M-mut patients (29.3 vs. 43.4 months, p = 0.04). The clinical outcomes in Exon20ins patients were not satisfactory compared to M-mut patients.