Currently, there is a trend towards an increasing use of liquid-based cytology (LBC) to diagnose non-small cell lung cancer. In this study, to detect epidermal growth factor receptor mutations, different molecular techniques were applied to LBC samples with and without laser capture microdissection (LCM). In 58 LBCs, DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after slide preparation and LCM of Papanicolaou-stained cells. The rate of mutant cases obtained by direct sequencing was discordant between CytoLyt-derived (10.3%) and LCM-derived (17.2%) DNA. However, the same mutant rate (17.2%) was achieved on the matched samples by high-resolution melting analysis, fragment and TaqMan assays. Thus, LCM and direct sequencing may be replaced by more sensitive non-sequencing methods directly performed on CytoLyt-derived DNA, an easier and faster approach to improve epidermal growth factor receptor testing standardisation on LBCs.