EGFR G724S has been described to mediate resistance to first- and third-generation EGFR tyrosine kinase inhibitors (TKIs). In vitro experiments have provided compelling evidence that G724S retains sensitivity for afatinib. Nevertheless, limited data have reported the clinical efficacy of afatinib in patients with NSCLC harboring G724S mutation.We identified 52 patients with NSCLC with EGFR G724S from an inhouse database and comprehensively profiled their concurrent mutation statuses. Treatments and clinical outcomes were also collected.Of 52 G724S-positive patients, 39 harbored concomitant EGFR exon 19 deletion (19del), and all 37 of the 39 patients who had available clinical data were detected with a G724S mutation after receiving EGFR TKIs. A rare variant of 19del E746_S752delinsV co-occurred with G724S the most frequently (n = 29), whereas 7 of 10 patients with concomitant EGFR exon 20 mutation were TKI treatment naive. S768I was the most common mutation in exon 20 (n = 7). One patient harbored a concomitant EGFR exon 21 mutation, and two lacked co-occurring EGFR mutations. A total of 23 patients provided valid clinical outcome data, of whom eight were treated with afatinib after the emergence of G724S, whereas 15 received non-afatinib treatment (alternative EGFR TKI, chemotherapy, or best supportive care). The disease control rate in afatinib-treated patients (n = 8) reached 100% with a median progression-free survival of 4.5 months, significantly longer than that of non-afatinib-treated (n = 15, 1.7 mo, hazard ratio [HR] = 0.32, p = 0.037) and alternative EGFR TKI-treated (n = 11, 1.8 mo, HR = 0.28, p = 0.042) patients. In the subset who had progressed on osimertinib, afatinib also yielded a superior progression-free survival (6.2 mo) than non-afatinib therapies (1.0 mo, HR = 0.04, p = 0.005) and alternative EGFR TKIs (1.8 mo, HR = 0.06, p = 0.033). Analysis of acquired mutations at afatinib progression revealed re-emergence of EGFR T790M or MET amplification as the potential mechanism of afatinib resistance.EGFR G724S emerges as a resistant mutation against EGFR TKI preferentially in the context of a rare variant of 19del, whereas it might mediate differential mechanisms in the context of exon 20 mutation. We also found that afatinib could be a potential therapeutic option for patients with NSCLC with G724S.© 2021 The Authors. Published by Elsevier Inc. on behalf of the International Association for the Study of Lung Cancer.