The genetic abnormalities involved in the pathogenesis of gallbladder carcinoma (GBC) remain unclear. Microsatellite instability (MSI) has been described in many carcinomas, but little is known about the significance of mismatch repair in gallbladder carcinogenesis. Additionally, methylation status of long interspersed element-1 (LINE-1), a surrogate marker of global DNA methylation, has defined distinct subsets of other cancer types but has not been explored in GBC. Immunohistochemical expression of MSH2, MSH6, MLH1, and PMS2 and LINE-1 mRNA in situ hybridization was evaluated in 67 primary and 15 metastatic GBCs from 77 patients. Amplification of human epidermal growth factor receptor 2 (HER2) was evaluated by fluorescence in situ hybridization. Genotyping for 24 genes involved in carcinogenesis was performed using a multiplex PCR-based platform. MSI was present in 6 of 77 GBCs (7.8 %). Loss of MSH2/MSH6 was detected in five cases and loss of MLH1/PMS2 in one case. MSI status was not associated with Lynch syndrome, tumor grade, extracellular mucin, or tumor-infiltrating lymphocytes. There was no significant difference in mean overall survival of patients with and without MSI. Strong LINE-1 staining was identified in none of the GBC with MSI and in 36 of 69 (52 %) of those without MSI (p = 0.005), suggesting that LINE-1 in the former cohort was hypermethylated. All MSI tumors were negative for HER2 amplification, and TP53 and NRAS mutations were only found in GBC without MSI. MSI was identified in a minority of GBC cases. The strong correlation between global DNA methylation as measured by LINE-1 and loss of mismatch repair proteins suggests that methylation may account for the loss of these proteins. These hypermethylated tumors appear to represent a genetically unique cohort of gallbladder neoplasms, and the data suggests that demethylating agents may have a therapeutic value in this class of tumors.