We investigated the frequency of microsatellite alteration and their impact on survival in head and neck squamous cell carcinoma patients from an endemic betel quid chewing area. We collected 116 head and neck squamous cell carcinoma specimens along with corresponding surgical margins which were confirmed by pathological examination. Ten oligonucleotide markers were chosen for the assessment of microsatellite alteration. The specimens were amplified by polymerase chain reaction followed by automatic fragment analysis. There were 44 specimens (37.9%) with microsatellite instability (MSI) in at least one marker while more than half of the specimens (n = 68, 58.6%) had loss of heterozygosity (LOH) in at least one marker. Though MSI/LOH was not correlated with the survival of head and neck squamous cell carcinoma patients, presence of MSI in the tumor-free surgical margins was associated with local recurrence (odds ratio: 15.14; 95% confidence interval: 6.451 ~ 35.53; P < 0.001). Genomic assessment of surgical margin can help surgeons to identify head and neck squamous cell carcinoma patients who are at risk of developing local recurrence in a betel quid-prevalent region.