BACKGROUND: Hepatobiliary scintigraphy (HBS) is a useful diagnostic imaging technique that uses radiotracers to evaluate the function of the gallbladder (GB) and biliary system. In segmented GB, some HBS images reveal a discordant GB boundary as compared to anatomical images. AIM: To evaluate the characteristics of HBS in segmented GB and determine the clinical relevance according to HBS characteristics. METHODS: A total of 268 patients with chronic cholecystitis, gallstones, or biliary colic symptoms who underwent HBS between 2011 and 2020 were enrolled. Segmented GB was defined as segmental luminal narrowing of the GB body on computed tomography (CT) or magnetic resonance (MR) images, and HBS was examined 1 mo before or after CT or MR. Segmented GB was classified into 3 types based on the filling and emptying patterns of the proximal and distal segments according to the characteristics of HBS images, and GB ejection fraction (GBEF) was identified: Type 1 was defined as a normal filling and emptying pattern; Type 2 was defined as an emptying defect on the distal segment; and Type 3 was defined as a filling defect in the distal segment. RESULTS: Segmented GB accounted for 63 cases (23.5%), including 36 patients (57.1%) with Type 1, 18 patients (28.6%) with Type 2, and 9 patients (14.3%) with Type 3 emptying pattern. Thus, approximately 43% of HBS images showed a discordant pattern as compared to anatomical imaging of segmented GB. Although there were no significant differences in clinical symptoms, rate of cholecystectomy, or pathological findings based on the type, most gallstones occurred in the distal segment. Reported GBEF was 62.50% ± 24.79% for Type 1, 75.89% ± 17.21% for Type 2, and 88.56% ± 7.20% for Type 3. Type 1 showed no difference in reported GBEF compared to the non-segmented GB group (62.50% ± 24.79% vs 67.40% ± 21.78%). In contrast, the reported GBEF was higher in Types 2 and 3 with defective emptying and filling when compared to Type 1 (80.11% ± 15.70% vs 62.57% ± 24.79%; P = 0.001). CONCLUSION: In segmented GB, discordance in the filling patterns detected by HBS and anatomical imaging could lead to misinterpretation of GBEF. For this reason, clinicians should be cautious when interpreting HBS results in patients with segmented GB.