Afatinib, a second-generation, irreversible pan-HER inhibitor, shows better suppression of T790M-positive lung cancer cells than gefitinib in preclinical studies. However, whether the effect of afatinib on T790M acquisition differs from that of gefitinib when used clinically as first-line therapy remains unclear. To reaffirm the preclinical efficacy of afatinib on T790M-positive lung cancer cells, H1975 cells and established PC-9 cells resistant to gefitinib and erlotinib by T790M were used. In total, 398 patients with second biopsy at progression with stage IIIB/IV non-small cell lung cancer with EGFR mutation, treated with afatinib or gefitinib as first-line therapy, were retrospectively reviewed. Propensity score matching was used to balance covariates. Afatinib inhibited the growth of lung cancer cells with low T790M allele frequencies, which are resistant to gefitinib, but not those with high T790M allele frequencies. Afatinib and gefitinib showed similar efficacy in terms of progression-free survival (PFS) (11.5 vs 13.4 months, P = .08) and overall survival (OS) (29.3 vs 28.5 months, P = .76). T790M patients had better PFS and OS than those without T790M. There was no significant difference in the cumulative T790M acquisition ratio over time between afatinib and gefitinib (48.8% vs 59.3%, P = .317). The median time to acquire T790M was 12.9 months for afatinib and 15.7 months for gefitinib (P = .342). Although afatinib inhibited the growth of lung cancer cells with low T790M allele frequencies in preclinical studies, this could not be translated into clinical efficacy in terms of lowering the rate or delaying the time of T790M acquisition.