Background: Leptomeningeal metastasis (LM) indicates an unfavorable prognosis in non-small cell lung cancer (NSCLC). Due to the blood-brain barrier, plasma is not an ideal medium to evaluate osimertinib resistance mechanisms in patients with LM. This study aimed to use cerebrospinal fluid (CSF) as a source of ctDNA for characterizing molecular features and monitoring intracranial responses from patients with leptomeningeal metastases who progressed on osimertinib. Methods: From Jan 2020 to Nov 2022, 65 patients with advanced NSCLC, including 32 osimertinib-treated patients, 6 other TKI-treated patients, and 27 treatment-naïve patients, in Beijing Tiantan Hospital were enrolled in this study. CSF obtained via lumbar puncture was used for targeted sequencing and cytologic analysis. In parallel, paired plasma from 35 patients was also subjected to targeted sequencing. Results: Among 65 patients, the overall positive rate of CSF ctDNA was 77% (50/65), 94% (34/36) in patients with LM, and 55% (16/29) in patients with brain parenchymal metastases (p = 0.0002). CSF ctDNA analysis had a higher consistency with the cytologic diagnosis of LM than imaging. Among the patients cytologic diagnosed with LM, 94% (32/34) of the patients had positive CSF ctDNA analysis at the same time, while the positive diagnostic rate of imaging was only 39% (13/33). EGFR alteration was found in 97% (29/30) CSF of patients with LM who progressed on Osimertinib, including L858R (67%), exon19 deletion (17%), T790M (10%), L861Q (7%), G719X (7%) and L747X (7%). Other recurrent genes discovered in the CSF were TP53 (73%), PIK3CA (43%), ERBB2 (33%), POLE (33%), ALK (27%), BRCA2 (27%), BRAF (23%), BRCA1 (23%), RB1 (20%) and PALB (20%). In the 35 paired CSF-plasma pairs, more alterations were uniquely found in CSF than in plasma (71% vs 17%), and only 12% of alterations were shared. Serial surveillance of CSF ctDNA in 7 cases of LM patients treated with osimertinib found that the VAF of CSF ctDNA showed a highly consistent trend with the tumor cell fraction of cytology. Conclusions: For NSCLC patients with LM, liquid biopsy of CSF is more sensitive than plasma to characterize osimertinib-resistant mechanisms and guide follow-up treatment. Serial surveillance of CSF ctDNA dynamics may reflect the intracranial tumor responses, which still needs to be validated in a large sample size.