Accurate identification of the intersegmental plane is essential in segmentectomy, and Indocyanine Green (ICG) assists in visualizing lung segments. Various factors, including patient-related, intraoperative, and technical issues, can influence boundary delineation. This study aims to assess the rate of unsuccessful intersegmental identification and identify the contributing factors. We analyzed cases of lung segmentectomy from April 2020 to March 2023, where intraoperative ICG was intravenously administered during robot-assisted or video-assisted thoracoscopic surgery. Cases where fluorescence extended beyond expected boundaries within 30 s were classified as the “unclear boundary group”. This group was then compared to the “clear boundary group”. The study encompassed 111 cases, 104 (94%) of which were classified under the “clear boundary group” and 7 (6%) under the “unclear boundary group”. The “unclear boundary group” had a significantly lower DLCO (15.7 vs. 11.8, p = 0.03) and DLCO/VA (4.3 vs. 3.0, p = 0.01) compared to the “clear boundary group”. All cases in the “unclear boundary group” underwent lower lobe segmentectomy. ICG administration effectively outlines pulmonary segments. Challenges in segment demarcation may occur in cases with low DLCO and DLCO/VA values, particularly during lower lobe segmentectomy.