Abstract Background: To secure negative surgical margins is the primary goal of surgical cancer treatment. However, tumor recurrence sometimes occurs even with histologically negative surgical margins. In order to detect submicroscopic residual cancerous cells intra-operatively, a time saving molecular biological approach is required. Methods: We established an innovative rapid Quantitative methylation PCR (QMSP) assay, which consists of remarkably time-minimized DNA extraction, bisulfite treatment and QMSP quantification. In order to assess the feasibility of completing the rapid assay during a surgical procedure, 10 serial specimens of primary head and neck cancers were evaluated by both rapid QMSP and conventional QMSP. Results: The DNA product quality of the most time saving procedure was as good as that of a conventional procedure. In the QMSP assay, target gene methylation and reference gene methylation were equally detected by both rapid and conventional methods. Finally, two frequently methylated genes in head and neck cancers, homeobox A9 (HOXA9) and endothelin receptor type B (EDNRB) were analyzed in 10 head and neck tumors, margin tissues and normal tissues. Relative QMSP values of rapid and conventional QMSP were quite similar in almost all tumors, margins and normal tissues. The average total time for the rapid QMSP procedure was less than 3 hours. Although postoperative follow-up periods are less than a year, local tumor recurrence occurred in one case whose margin tissue was QMSP positive. Conclusion: This innovative rapid QMSP yields acceptable levels of methylation detection and can be completed during the operative window. The procedure promises to become a useful clinical tool, if the methylated surgical margins are revealed to correlate with the local tumor recurrence in a larger cohort. Citation Format: Masamichi Hayashi, Rafael Guerrero-Preston, Zubair Kahn, Xiufeng Li, Julie Ahn, Marla Goldsmith, Christina Michailidi, Wayne Koch. Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2014-3837