Background <p>This study aimed to assess the ability of FoundationOne Liquid CDx (F1 Liquid), a blood-based comprehensive genomic profiling test introduced in Japan in 2021, to detect the H3K27M mutation in patients with diffuse midline glioma using a nationwide real-world database in Japan.</p> Methods <p>We identified patients with IDH-wildtype diffuse gliomas in midline locations registered in the Center for Cancer Genomics and Advanced Therapeutics database from June 2019 to April 2024. Detection rates of the H3K27M mutation and other major variants were compared across two blood-based comprehensive genomic profiling tests, F1 Liquid and Guardant360 CDx, and three tissue-based, FoundationOne CDx (F1 CDx), NCC Oncopanel, and GenMine TOP. Predictors of H3K27M mutation detection were analyzed using logistic regression.</p> Results <p>We identified 114 patients with diffuse gliomas located in the midline. Among these, 31.6% underwent F1 Liquid testing, which had only a 2.8% detection rate for the H3K27M mutation. In contrast, F1 CDx testing had a significantly higher detection rate of 92.2% (<i>p</i> &lt; 0.01). Not undergoing F1 Liquid testing was the only independent predictor of H3K27M detection (odds ratio, 267; 95% confidence interval, 44.4–5250; <i>p</i> &lt; 0.01). F1 Liquid also showed low detection rates for TP53 (2.8%), PDGFRA (0%), and NF1 (2.8%).</p> Conclusion <p>Our findings indicate a critical limitation of F1 Liquid in detecting the H3K27M and other significant mutations in diffuse midline glioma.</p>