Abstract Background Assessing circulating cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) has been proposed as a promising alternative to tissue biopsy. Advances in cfDNA sequencing have further underscored the potential of CSF liquid biopsies. CSF is routinely collected for cytologic evaluation at diagnosis, and at recurrence in both pediatric and adult central nervous system (CNS) tumors. Methods As part of a pilot study, CSF specimens were prospectively collected from seven pediatric patients with primary CNS malignant tumors. When possible, CSF was collected fresh and/or from processed cytology specimens. Low-pass whole genome sequencing (LP-WGS) and next-generation sequencing (NGS) using a custom targeted sequencing panel were performed on the specimens to identify copy number alterations (CNAs), detect mutations, and estimate circulating tumor DNA (ctDNA) fractions. Results were compared with matched tumor tissue molecular profiles and corresponding imaging findings. Results Abnormalities in cfDNA were detected in four patients. Sequencing of CSF cytology supernatants demonstrated the presence of circulating tumor DNA with characteristic CNAs and mutations that matched what was seen the tumor tissue as well as the fresh CSF specimens. These studies also revealed tumor heterogeneity and genomic evolution over time. Conclusion This study demonstrates the feasibility of utilizing routinely discarded supernatants from CSF cytology specimens for LP-WGS and targeted NGS. Our approach optimizes the use of CSF that may be limited in pediatric patients as a reliable source for liquid biopsy-based genomic studies. Future research will be necessary to optimize the methodology to enable clinical implementation. Key points Combined low-pass whole genome sequencing (LP-WGS) and targeted next-generation sequencing (NGS) can detect circulating tumor DNA (ctDNA) in cerebrospinal fluid (CSF) cytology supernatant samples. Our approach transforms clinically discarded specimens into a valuable and reliable source for liquid biopsy. LP-WGS and targeted NGS of CSF cytology-derived cell-free DNA (cfDNA) is more sensitive than CSF cytology for tumor detection at initial diagnosis and during surveillance. Importance of Study This report highlights the feasibility of utilizing supernatants from cerebrospinal fluid (CSF) cytology specimens that would normally be discarded for diagnosis and follow up for patients with pediatric primary central nervous system (CNS) tumors, using the LBSeq4Kids combined low-pass whole genome sequencing (LP-WGS) and targeted next-generation sequencing (NGS) liquid biopsy platform. It optimizes the use of limited clinical specimens and maximizes diagnostic and research potential, yet does not pose additional risk to patients by utilizing routinely collected cytology specimens. Identification of copy number alterations (CNAs) and DNA sequence variants detected in CSF-derived cell-free DNA from patients with high grade glioma and embryonal tumors can augment tissue-derived molecular analyses. CSF liquid biopsy approaches have the potential to transform clinical practice for improved diagnosis, risk stratification, and disease surveillance.