Background In gallbladder cancer (GBC), extrahepatic bile duct (EHBD) resection is selectively performed if gross direct extension or microscopic involvement of the cystic duct margin (CDM) is detected. Although CDMis usually sent for frozen biopsy intraoperatively in most centers, there are no studies regarding the routine use of CDM frozen biopsy irrespective of the tumor location and paucity of literature regarding the impact ofCDM status on recurrence-free and overall survival inGBC. The presence of obstructive jaundice in GBC usually indicates the involvement of EHBD or cystic duct-bile duct junction. The present study aimed to analyze the necessity of routineCDM frozen biopsy inpatients with resectable GBC withoutjaundice, regardless of the tumor location. The impact of positive CDM on survival was also evaluated. Methods This retrospective observational case-control study was conducted from May 2009 to March 2021 and included 105 patients with resectable GBC without macroscopic EHBD infiltration andjaundice. Patients were divided into CDM-negative (n=91) and CDM-positive (n=14) groups. Propensity score matchingwas performed for variables such asperformance status, tumor size, tumor-node-metastasis (TNM) stage, and adjuvant chemotherapy. After propensity score matching, 27 patients (CDM-negative=13, CDM-positive=14) were included. The primary outcome was to analyze the role of routine CDM frozen biopsy regardless of tumor location, and secondary outcomes were to study the impact of positive CDM status on survival andevaluate predictive factors for CDM positivity. A subgroup analysis was conducted to assess clinicopathologic characteristics and outcomes of the anatomical location of the tumor. Results Of 105 patients, 91 had negative CDM, and 14 had positive CDM. Among 14 patients with positive CDM, only one patient had a tumor in the fundus/body, and the remaining had a tumor involving the neck. All CDM-positive patients underwent bile duct excision with hepaticojejunostomy. Common bile duct (CBD) involvement was present in 50% of patients with positive CDM in the final histopathological examination. In the matched population, patients with positive CDM had a significantly higher rate of neck tumors (p=0.001). Recurrence-free survival (24 vs. 12 months, p=0.30) and overall survival (24.5 vs. 20 months, p=0.417) were comparable between CDM-negative and CDM-positive groups, respectively. On multivariate analysis, preoperative and intraoperative tumor location were independent predictive factors for CDM positivity. On subgroup analysis, 30 patients had tumor involving the neck of the gallbladder, and the remaining 75 had at the fundus and body of the gallbladder. Neck tumors had inferior recurrence-free survival (17 vs. 30 months, p=0.012) and overall survival (24 vs. 36 months, p=0.048) compared to non-neck tumors. Conclusions Routine use of CDM frozen analysis in patients with resectable GBC without jaundice, regardless of tumor location, can be avoided. Itcan be selectively preferred in patients with GBC involving the neck since tumor location is found to be an independent predictive factorfor CDM positivity. Positive CDM has comparable survival outcomes to negative CDM, providing a similarR0 resection rate and tumor stage.However, neck tumors have aworse prognosis than non-neck tumors.