Background: Liquid biopsy of cerebrospinal fluid (CSF) has been approved to be a promising strategy for molecular pathology testing in patients with lung cancer. Numerous studies showed that CSF-derived DNA can recapitulate the molecular characteristics of metastatic lung cancer. However, differences in mutation detection rate and the overall mutational profile between cell pellet DNA and supernatant cell-free DNA (cfDNA) from CSF in patients with lung cancer have rarely been reported. Methods: Matched CSF cell pellet and supernatant sample sets were selected from 51 lung cancer patients in our sequencing center. CSF was centrifuged (1000g, 10 min) to separate the cell pellet and supernatant components. Cellular DNA and cfDNA were extracted and underwent library preparation procedures for next-generation sequencing respectively. Cut adapter, mapping, fusion calling and cnv calling were performed for sequencing data analysis. Results: At the sample level, gene mutation could be detected in both CSF cell pellet and cfDNA, and the positive detection rate of gene mutation was consistent. The positive percent agreement (PPA) of CSF cfDNA sequencing compared with CSF cell pellet DNA was 93%. At the genetic variation level, 223 gene mutation sites were detected, and the positive rate of gene mutation in the CSF cfDNA (32.51%) was significantly higher than that in the CSF cell pellet (10.74%, p＜0.01). Additionally，the variation rate of less than 1% was defined as the low-frequency mutation range. Results showed that variant allele frequency (VAF) in CSF cfDNA were remarkably higher than those in the CSF cell pellet in both low-and high-frequency mutation ranges (p＜0.01, p＜0.01). Conclusions: Both cell pellet DNA and cfDNA isolated from CSF can be used for molecular subtyping of lung cancer. Given that CSF is inevitably contaminated with blood cells in clinical procedures, the dynamic range of tumor cells in CSF could be quite large. CSF cfDNA better recapitulate the mutational status and is recommended to consider first for liquid biopsy in advanced lung cancer with brain metastases or suspicious brain metastases. Key words: Next-generation sequencing; CSF; cell pellet DNA; cfDNA; advanced lung cancer.