BackgroundA high recurrent rate of oral squamous cell carcinoma (OSCC) is a major concern in head and neck cancer treatment. The study of the genetic mutation landscape in recurrent OSCC may provide information on certain mutations associated with the pathobiology and treatment response of the OSCC.AimWe investigated the mutation landscape of matched pretreatment and recurrent tumors to understand the influence of genetic mutations on the pathobiology and clinical outcomes in OSCC.Methods and ResultsWe sequenced 33 formalin‐fixed paraffin‐embedded (FFPE) recurrent tumors, primary tumors, and primary tumors before recurrence that matched the recurrent tumors collected from Rajavithi Hospital during 2019–2021. We identified recurrent mutations from these samples by the Oncomine Ion Torrent‐based next‐generation sequencing on the 517 cancer‐associated gene panel. From the results, we found that the most commonly mutated gene in the cohort is a histone methyltransferase KMT2D (54.55%), implicating that aberrance in epigenetic regulation may play a role in oral cancer tumorigenesis. Functional protein association network analysis of the gene frequently mutated in the recurrent tumors showed enrichment of genes that regulate the cancer cell cycle, that is, MRE11A, CDKN2A, and CYLD. This finding was confirmed in the primary‐recurring matched pair. We found that recurrent tumors possess a small but recurring group of genes, with presumably the subclonal mutations driving the recurrence of the tumor, suggesting that the recurrent disease originated from a small fraction of the cancer cell that survives standard treatment. These genes were absent in the primary tumor with a good response to the standard treatment. On the other hand, we found an enrichment of DNA repair genes, namely ATR, BRCA1, BRCA2, RAD50, and MUTYH, in nonrecurrent tumors suggesting that the mutations in the DNA repair pathway may at least partially explain the different response to the standard treatment.ConclusionsOur pilot study identified pathways of carcinogenesis in oral cancer and specific gene sets that indicate treatment responses and prognoses in this group of patients.