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Introduction Cytological samples obtained by endobronchial ultrasound (EBUS) are capital for diagnosis, staging and molecular profile in non-small cell lung carcinoma (NSCLC). Objective To assess the success rate of complete, partial and individual of molecular analysis in samples obtained by EBUS-guided transbronchial needle aspiration (TBNA) and/or by oesophageal ultrasound-guided fine needle aspiration with an echobronchoscope (EUS-B-FNA) in patients with NSCLC. Methods Prospective study including 90 patients with non-squamous NSCLC, or non-smoking squamous. Cytological samples were classified into two groups. Group 1: PEN membrane slide and/or cell blocks for the determination of mutations of EGFR, KRAS, ERBB2 and BRAF. Group 2: silane coated slides or cell blocks for rearrangements of ALK, ROS1 and MET amplification. Results The success rate was 78.6% for 4 molecular alterations (EGFR, KRAS, ALK and ROS1), and 44% for 7 determinations. The individual success rate for EGFR was 97%, KRAS 96.3%, ALK 85%, ROS1 82.3%, ERBB2 71.4%, BRAF 67.7% and MET 81.1%. There were no significant differences (p = 0.489) in the number of molecular analyses (1–3 vs. 4) in group 1, depending on the types of samples (cell block vs. PEN membrane slide vs. cell block and PEN membrane slide). Conclusions In patients with NSCLC, the cytological material obtained by ultrasound-guided needle aspiration is sufficient for individual and partial molecular analysis in the vast majority of cases. Membrane slides such as cell blocks are valid samples for molecular analysis.