Although somatic mutations were explored in depth, limited biomarkers were found to predict the resistance of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Previous studies reported N6-methyladenosine (m6A) levels regulated response of EGFR-TKIs, whether the germline variants located in m6A sites affected resistance of EGFR-TKIs is still unknown.Non-small cell lung cancer (NSCLC) patients with EGFR-activating mutation were enrolled to investigate predictors for response of EGFR-TKIs using a genome-wide-variant-m6A analysis. Bioinformatics analysis and series of molecular biology assays were used to uncover the underlying mechanism.We identified the germline mutation USP36 rs3744797 (C>A, K814N) was associated with survival of NSCLC patients treated with gefitinib (median PFS: CC vs. CA, 16.30 vs. 10.50 months, P<0.0001, HR=2.45) and erlotinib (median PFS: CC vs. CA, 14.13 vs. 9.47 months, P=0.041, HR=2.63). Functionally, the C>A change significantly upregulated USP36 expression by reducing its m6A level. Meanwhile, rs3744797_A (USP36 MUT) was found to facilitate proliferation, migration, and resistance to EGFR-TKIs via upregulating MLLT3 expression in vitro and in vivo. More importantly, MLLT3 and USP36 levels are tightly correlated in NSCLC patients, which were associated with prognosis of patients. Mechanistically, USP36 MUT stabilized MLLT3 by deubiquitinating MLLT3 in nucleoli and consequently activating its downstream signaling (HIF-1α and Snai). Furthermore, inhibition of MLLT3 alleviated USP36 variant-induced EGFR-TKIs resistance in EGFR-mutant NSCLC.These findings characterized rs3744797 as an oncogenic variant in mediating EGFR-TKIs resistance and tumor aggressiveness through deubiquitinating MLLT3, highlighting the variant as a predictive biomarker for EGFR-TKIs response in NSCLC.