T790M mutations inEGFR-mutated non-small cell lung cancer (NSCLC) account for nearly 50% of acquired resistance mechanisms to EGFR-TKIs. Earlier studies suggested that tumor T790M could also be detected in TKI-naïve EGFR-mutated NSCLC. The aim of the study is to assess the prevalence and clinical significance of quantification of tumor pre-treatment T790M subclones.We analyzed 366 EGFR-mutated NSCLC patients of the real-life IFCT Biomarkers France study with available pre-treatment formalin-fixed paraffin-embedded (FFPE) tumor DNA before treatment by first/second-generation EGFR-TKI. We used ultra-sensitive Droplet Digital Polymerase Chain Reaction (ddPCR) QX200 (BIO-RAD®, Hercules, CA, USA). All samples were tested in duplicate.ddPCR identified T790M in 19/240 specimens (8%). T790M-positive and T790M-negative populations were not different for clinical baseline characteristics. T790M Variant Allele Frequency (VAF) was > 0.01% <0.1%, > 0.1% <1%, > 1% <10%, and >10% in five (26.3%), six (31.6%), six (31.6%), and two (10.5%) patients, respectively. T790M VAF was >0.1% in 11/13 (84%) patients with rapid (<3 months) or usual progression (3-20 months) compared to 0/3 with low progression (>20 months) (p = 0.02). In a Cox model, T790M mutation positivity was correlated with overall survival (OS) and progression-free survival (PFS) for 10% > VAF >1% (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.13-7.07, p = 0.03; HR=3.62, 95%CI 1.43-4.92, p = 0.007, respectively) and for VAF >10% (HR = 19.14, 95%CI 4.35-84.26, p < 0.001; HR = 17.89, 95%CI 2.21-144.86, p = 0.007, respectively).Ultra-sensitive detection of tumor T790M mutation concerned 8% of EGFR-mutated TKI-naïve NSCLC patients and has a negative prognostic value only for T790M VAF over 1%.