The aim of this study was to investigate the association between microsatellite alteration in the surgical margins and local recurrence of oral cavity squamous cell carcinoma patients. Surgical specimens confirmed by pathological examination and corresponding surgical margins were collected from 120 oral cavity squamous cell carcinoma patients. Ten microsatellite markers were examined in the tumor specimens and paired surgical margins, which proved to be negative on pathological assessment. The specimens and surgical margins were amplified by polymerase chain reaction followed by computerized analysis. Forty-two specimens (35.0 %) with microsatellite instability (MSI) in at least one marker were found, and more than half of the specimens (n = 73, 60.8 %) had loss of heterozygosity (LOH) in at least one marker. Although MSI and LOH were not associated with the prognosis of oral cavity squamous cell carcinoma patients, presence of MSI in the tumor-free surgical margins increased the risk of local recurrence (hazard ratio: 9.549; 95 % confidence interval: 4.143–22.01). Genetic analysis of tumor-free surgical margins is a useful tool for identifying oral cavity squamous cell carcinoma patients who are vulnerable to local recurrence.