Leptomeningeal metastasis (LM) are a severe complication of non-small cell lung cancer (NSCLC), and normally accompanied by poor prognosis. For the patients with targetable mutations, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the preferred treatment, but the acquired TKI resistance is inextricable. The aim of this study is to analyze the different gene mutation spectrum and mutation frequency of the cerebrospinal fluid (CSF) and plasma in NSCLC patients with LM, and screen out the drug-resistant mutations so as to guide the choice of treatment accurately.The paired CSF and plasma samples were collected from the NSCLC-LM patients with acquired TKI resistance. Next generation sequencing (NGS) was used to detect the gene variations of circulating tumor DNA (ctDNA).A total of 18 NSCLC patients with LM were collected. Of the basic mutations, 11 cases (61.11%) were EGFR, 6 cases (33.33%) were anaplastic lymphoma kinase (ALK), and 1 case (5.56%) was ROS proto-oncogene 1, receptor tyrosine kinase (ROS1). Tumor protein p53 gene (TP53) and mesenchymal-epithelial transition factor (MET) were the two most frequently accompanying mutated genes in CSF ctDNA. The detected mutation rate of CSF samples was higher than that of plasma samples (100.00% vs 66.67%, P=0.006), and the maximum allelic fractions were all higher in CSF than in plasma (P<0.001). Abundant single-nucleotide variations (SNV) and copy number variants (CNV) were detected in CSF, the amount of both of which were more than in blood. In addition, the CSF and plasma samples of patients treated with several TKIs had more SNV mutations than patients who received only a single TKI treatment.For the patients of NSCLC, ctDNA in CSF could reveal genomic alterations of LM more exactly and overally than it in plasma, thus could be an optimal source of liquid biopsy for guiding therapy, monitoring therapeutic effect, and predicting prognosis.