Purpose Routine histopathological examination after cholecystectomy for gallstones is performed despite the low rates of incidental findings of malignancy. The aim of this study was to assess predictive values of macroscopic examination of cholecystectomy specimens by surgeons in gallstone disease. Methods A prospective multi-center diagnostic study was carried out between December 2015 and March 2017 at four different centers. All patients undergoing cholecystectomy for gallstone disease were consecutively screened for eligibility. Patients whose ages are 18 to 80 years, and preoperative imaging findings without any pathology except cholelithiasis were included. The gallbladder was first evaluated macroscopically ex situ by two operating surgeons and rated as macroscopically benign (group S1), suspicious for a benign diagnosis (group S2), and suspicious for malignancy (group S3). Thereafter, a pathologist made a final histopathological examination whose results are grouped as chronic cholecystitis (group P1), benign or precancerous lesions in which only cholecystectomy is the adequate treatment modality (group P2), and carcinoma (group P3). Diagnostic accuracy of the surgeon's assessment to the histopathological examination was evaluated using sensitivity, specificity, positive and negative predictive values, and accuracy, and correlated by a kappa agreement coefficient. Results A total of 1112 patients were included in this trial. The specificity rates were 96.5%, 100%, and 98.7% for group S1-group S2, group S1-group S3, and group S2-group S3, respectively. Accuracy rates to detect malignancy were 100% and 95. 2% for group S1 and group S2, respectively. Kappa coefficient values were 1.0 and 0.64 for group S1-group S3 and group S2-group S3, respectively (p < 0.001 for both). Conclusion Assessment of the gallbladder specimen and selective histopathological examination may be adequate after cholecystectomy for gallstone diseases. Such a procedure would have the potential to reduce costs and prevent unnecessary loss of labor productivity without affecting patients' safety. However, higher number of patients in more centers is needed to confirm this hypothesis.