The aim of this study was to examine the microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in gallbladder tumors, which may provide experimental basis for the tumor occurrence and metastasis. Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain were used to study MSI and LOH of locus D17S396. Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of gene nm23H1. In our experiment, the frequency of genetic instability of malignant gallbladder tumors was 42.55%, which was higher than that of gallbladder adenomas, while there were no genetic instability occurred in chronic cholecystitis tissue. The frequency of LOH seemed higher with the deteriorism of gallbladder tumor. Among 47 gallbladder carcinomas, the frequency of LOH and MSI were different between different differentiation cases (P < 0.05), and the frequency of LOH in liver and lymph node metastasis cases was significantly higher than those without metastasis (P < 0.01). Moreover, the frequency of LOH was higher in stage Nevin IV and V when compared with stage I, II and III. However, the frequency of MSI showed contrary correlation with some clinicopathologic characteristics. The expression of nm23H1 in gallbladder carcinoma, gallbladder adenoma and chronic cholecystitis tissue were different (P < 0.05). The case with lymph node metastasis showed significantly lower nm23H1 expression than those without lymph node metastasis (P < 0.01). Nevin stage IV and V also exhibited lower nm23H1 expression levels compared with stage I, II and Ill. Furthermore, there was no difference in nm23H1 protein expression intensity analyzed by computer imaging techniques. In gallbladder carcinomas, the positive frequency of nm23H1 protein in LOH positive group was lower than that of LOH negative group (P < 0.05). The results indicated that the genetic instability of nm23H1 gene might be implicated in pathogenesis and progression of gallbladder tumor. Both MSI and LOH of nm23H1 gene controlled the development of gallbladder tumor independently in different paths. MSI may be an early stage molecule marker of gallbladder carcinoma. LOH may be molecule marker for the deteriorism of gallbladder tissue, which could inhibit the expression of nm23H1 in local tissue of gallbladder carcinoma and endow it with high aggressive and poor prognosis. Increasing the amount of nm23H1 protein expression could effectively restrain gallbladder carcinoma metastasis and improve prognosis of patients.