Aberrant DNA methylation of a CpG site is among the earliest and most frequent alterations in various tumors including gastric carcinoma. The aim of this study is to detect tumor-associated aberrant hypermethylation of the p16 gene from 60 gastric tumor and corresponding normal tissues using a seminested methylation-specific PCR (MSP). The results indicated that hypermethylation of the p16 gene could be detected in 80% (48/60) of the gastric tumor samples from the first PCR. However, the frequency increased significantly to 86.7% (52/60) of the gastric tumor samples after the second PCR. These results show that this technique increases the sensitivity of detecting p16 hypermethylation from tumor samples. Furthermore, the aberrant methylation of p16 was observed in all of the stages, confirming that this epigenetic alteration is an early event during gastric carcinogenesis. Clinicopathologic parameters such as age, sex, and histological differentiation of GC were not significantly associated with the methylation status.