Background: Genomic profiling of cerebrospinal fluid (CSF) could be used to detect actionable mutations to guide the clinical treatment of NSCLC patients with central nervous system (CNS) metastases. Examining the performance of CSF samples in a real-world setting can further confirms the potential of CSF in genotyping for guiding therapy in clinical practice. Methods: A total of 1097 samples were collected from 773 treated NSCLC patients with CNS metastases in a real-world setting, including 117(10.67%) CSF samples, 287(26.16%) tissue samples and 693(63.17%) plasma samples. All samples were subjected to the targeted next-generation sequencing of 1021 cancer-relevant genes. Results: Of these 1097 treated samples, somatic alterations were identified in 112 (95.72%) of the CSF samples, comparing with 287 (100%) of tumor tissue samples and 592 (85.43%) of plasma. Among the tumor tissue samples, 242 were non-intracranial tissues, which could not reveal the unique genetic profiles of intracranial metastases. The median of maximal somatic allele frequency of CSF samples (72.35%) was significantly higher than those of plasma (1.30%) and tumor tissues (37.30%) (all p<0.001). In the thirty-two pairs CSF and plasma samples tested simultaneously, 442 alterations were detected, of which 377 were detected in CSFs and 92 in plasma. 27 alterations could be detected in both plasma and CSF, 65 were not detected in CSFs and 350 were not in plasma, the same alterations were 10.91% (27/442). For SNV or InDel, 220 mutations were detected, of which 155 were detected in CSFs and 89 in plasma, the same mutations were 10.91% (24/220). For CNVs, 216 CNV alterations were detected, of which 216 were detected in CSFs and only one in plasma. We compared actionable mutation of these 1097 treated samples to further analyze the detection capability of actionable mutations of CSF samples in this real-world setting (Table). Compared with plasma, the detection rates of all actionable mutation and actionable EGFR in CSF were significantly higher than those in plasma samples (93.16% vs. 53.97% for all actionable mutation, 83.76% vs. 39.54% for EGFR, all p<0.001).Conclusions: This real-world large cohort study verified that CSF had higher sensitivity than plasma for identifying actionable mutations. In the process of multiple comparison, it can be seen that CSF is better than plasma in detecting alterations, especially in detecting CNV alteration. CSF can be used as a substitute in genomic profiling for NSCLC patients with CNS metastases when there is no intracranial tumor tissue.