Microsatellite instability (MSI)-mutator phenotype variably targets microsatellite-like sequences in coding regions of cancer-related genes. Intratumor histological heterogeneity of gastric carcinoma with MSI was evaluated and found to be linked with the topographical distribution of MSI-associated mutations. One hundred fifty tumor sites derived from 51 gastric cancer patients were microdissected with respect to histological and topographical clonality. We found 11 gastric carcinomas with a high frequency of MSI, which were characterized by marked intratumor genetic heterogeneity arising from the progressive MSI-phenotype that was associated with frameshift mutations on multiple cancer-related genes. The 11 MSI-tumor cases manifested the MSI-phenotype in 34 of 36 tumor sites tested, but not in the remaining 2 sites. Most (88.2%, 30 of 34) MSI-positive sites and most (96.2%, 25 of 26) tumor sites harboring the frameshift mutations in transforming growth factor-β receptor type II gene exhibited intestinal-type histology, whereas the 2 MSI-negative sites were found to be of diffuse-type histology without accompanying frameshift mutations. In 2 of 5 cases harboring E2F-4 frameshift mutations, glandular structures of intestinal-type tumor were likely to be variably differentiated in relation to the extent of the mutation, i.e., the number of mutated alleles and the size of deleted or inserted base pairs. Overall, the intratumor histological heterogeneity of gastric carcinoma with MSI was associated with the progressive frameshift mutations in transforming growth factor-β receptor type II and E2F-4 genes.