BACKGROUND: Brain metastases (BM), including brain parenchyma metastases (BPM) and leptomeningeal metastases (LM), are devastating metastatic complications in advanced cancer patients. Next-generation sequencing (NGS) is emerging as a new promising tool for profiling cancer mutation, which could facilitate the diagnosis of cancer. This retrospective study aimed to investigate the molecular genetic characteristics of non-small cell lung cancer (NSCLC) patients with BPM and LM using NGS. METHODS: Cerebrospinal fluid (CSF) samples and paired plasma samples were collected from 37 patients of NSCLC-BM. We profiled genetic mutation characteristics using NGS from NSCLC-BM by comparing CSF circulating tumour DNA (ctDNA) with plasma ctDNA and primary tumour tissues. RESULTS: Among the 37 patients with NSCLC-BM, 28 patients had LM with or without BPM, while 9 patients only had BPM. Driver and drug-resistant mutations in primary tumours with LM included: L858R (10, 35.7%), 19del (6, 21.4%), L858R+ (1, 3.6%), L858R+S768I (1, 3.6%), (2, 7.1%), (1, 3.6%), negative (5, 17.9%), and unknown (2, 7.1%). In patients with NSCLC-LM, the detection rate and abundance of ctDNA in the CSF were significantly higher than those in paired plasma. The main driver mutations of NSCLC-LM remained highly consistent with those of the primary tumours, along with other unique mutations. Circulating tumour DNA was negative in the CSF samples of BPM patients. Patients with BMP had a higher ratio of 19del than L858R mutation (55.6% vs 11.1.%), whereas NSCLC patients with LM had a higher ratio of L858R than 19del mutation (50.0% vs 25.0%). Most patients with positive plasma ctDNA results were male ( = 0.058) and in an unstable state ( = 0.003). CONCLUSION: Our study indicated that the CSF ctDNA detected by NGS may reflect the molecular characteristics and heterogeneity of NSCLC-LM. Timely screening of patients with NSCLC for CSF ctDNA, especially for patients with positive plasma ctDNA, may facilitate the early detection of LM. Furthermore, patients with the 19del may have a higher risk of developing BPM.