Aberrant 5′ CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor‐suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5′ CpG island hypermethylation of hMLH1, E‐cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation‐specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E‐cadherin and p16 methylation respectively, and 8 (13.1%) presented high‐frequency MSI (MSI‐H). Of the 8 MSI‐H patients, 5 presented hMLH1 methylation, whereas no low‐frequency MSI (MSI‐L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E‐cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers. Int. J. Cancer 83:309–313, 1999. © 1999 Wiley‐Liss, Inc.