Pleural effusion (PE) is commonly observed in advanced lung cancer patients. Cell-free total nucleic acid (cfTNA) isolated from cancer patient’s plasma has allowed non-invasive tumor genome analysis, however, there are very limited studies of detection and characterization of cfTNA in PE. Here we included 47 stage IV NSCLC patients with PE, who had lung cancer driver mutations tested on tumor tissue specimens at either diagnosis or during disease progression. The supernatant and cell pellet of each PE were evaluated for molecular profiles in parallel on ion torrent NGS platform. Somatic mutations were detected in 89.1% supernatant cfTNA, while they were only detected in 54.3% of cell pellets. The overall concordance rate between supernatants and FFPE cell pellets at the mutation level was 53.3%. By contrast, 41.7% and 5.0% of somatic alterations were only detected in supernatants and cell pellets, respectively. Furthermore, joint analysis of supernatants and cell pellets from PE showed a high concordance (88.3%) of variant detection with their respective tumor tissue specimens. Low frequency T790M mutations in three cases (0.29%, 0.41%, 1.56%) were only detected in supernatants, but not in the matched cell pellets or tumor tissues. In conclusion, pleural effusion derived cfTNA can effectively be used in clinical practice for molecular analysis by NGS, even in cases corresponding cell pellets or tumor tissues yield insufficient material.Copyright © 2020. Published by Elsevier Inc.