Germline mutations in MSH2, MLH1, E-cadherin and MutY (MYH) genes have been implicated in the occurrence of gastric cancer (GC). Epidemiological investigation was performed by recruiting patients with GC onset during 2002 in Jiangsu province, China. We identified suspected hereditary GC patients based on either the GC family history or GC onset at early ages. We have screened germline variations in 101 suspected hereditary GC patients at the coding sequences of MSH2, MLH1, E-cadherin and MYH genes with polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC) analysis and DNA sequencing. The result showed that about 40% of patients carried germline variations, predominantly with missense mutations. Of the variations detected are 2 base pair substitutions, c.53C > T and c.74G > A, which is predicted to generate missense mutations of p.Pro18Leu and p.Gly25Asp, respectively, and occurred at the same allele of MYH gene. The frequency of variant haplotype T/A in patients was higher than that in the control group (p = 0.021, odds ratio [OR] = 4.43, 95% confidence interval [95% CI] = 1.33-14.72). Difference in the frequency of the silent mutation p.Asn751Asn in E-cadherin gene was also found between patients and controls (p = 0.009, OR = 2.54, 95% CI = 1.30-4.95). Moreover, 6 types of variations were detected in MSH2 and MLH1 genes in 14 of 101 patients. Most of them occurred at exon7 of MSH2, frequently c.1168C > T, resulting in mutation of p.Leu390Phe. In summary, germline mutation at MSH2, MLH1, E-cadherin and MYH genes is a frequent event in the familial GC. They may form a genetic basis for the familial GC susceptibility in Chinese population.