Phenotyping non-small-cell lung cancer is becoming increasingly important with the advent of molecular testing. Tumours harbouring somatic mutations in the gene that encodes for the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) have been found to increase responsiveness to tyrosine kinase inhibitors. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for mediastinal node sampling. The available prospective data on EBUS-TBNA sample suitability for molecular profiling are currently limited. The aim of this prospective study was to evaluate the adequacy of EBUS-TBNA samples for EGFR and anaplastic lymphoma kinase (ALK) genetic mutation analysis in confirmed primary lung adenocarcinomas. We conducted a prospective analysis of 410 consecutive patients referred for EBUS-TBNA between 2010 and 2014. Rapid on-site cytological evaluation was not used. The samples were obtained using 21-gauge (21G) or 22G needles and were prepared as histopathological samples. A total of 91 samples were confirmed as lung adenocarcinomas and 80 of these samples were sent for EGFR mutation analysis. EBUS-TBNA had a diagnostic accuracy of 98.3% for malignancy. EGFR mutation testing was possible in 79/80 cases (98.75%). EGFR mutations were detected in 5/80 (6.3%) samples. ALK gene analysis, which became available during the study period, was requested and successfully performed in 21/21 samples (100%). The total combined genotyping success rate was 100/101 (99.0%). This UK study confirmed the high clinical utility of EBUS-TBNA samples processed as histopathological specimens for EGFR and ALK genotyping in primary lung adenocarcinoma. The needle gauge did not affect genotyping efficacy.