The malignant transformation that characterizes the development of Barrett esophagus-associated adenocarcinomas is a multi-step process in which genetic alterations in various tumor-associated genes accumulate. Defective mismatch repair (MMR) is the cause of microsatellite instability (MSI) pathway that characterizes a subset of gastrointestinal tumors and is specifically associated with tumor development within the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. The few studies that have assessed MMR defects in Barrett-associated adenocarcinomas have reached different results. We therefore assessed the expression of the MMR proteins MLH1 and MSH2 in a series of 59 Barrett adenocarcinomas and found a loss of MMR protein immunostaining in 2/59 (3%) tumors; one tumor showed a loss of MSH2 expression, the other tumor showed a loss of MLH1, and both tumors displayed an MSI-high phenotype. Our findings suggest that only a small subset of Barrett adenocarcinomas develop because of defective MMR, but demonstrate that MLH1 and MSH2 are the primary targets for defective MMR also in this tumor type.