Abstract Background If the depth of gallbladder malignant tumor (GBMT) invasion is deeper than the subserosa (ss), cholecystectomy is insufficient. In past reports that used endoscopic ultrasonography (EUS) to diagnose the depth of tumor invasion, it was difficult to diagnose GMBT invasion in the ss without a narrow or disrupted lateral hyperechoic layer (LHEL). Therefore, we developed a simple preoperative method to diagnose GBMTs with ss invasion. Methods Forty-nine GBMT patients who underwent both EUS and surgery were enrolled: 15 patients whose tumors invaded the mucosa (m) or muscularis propria (mp) were classified as the “shallow group”, and 34 patients whose tumors invaded the ss were classified as the “deep group”. The EUS findings were compared between the two groups. Results An irregular (narrow or thickened) LHEL was significantly more frequently observed on EUS in the deep group than in the shallow group. The diagnosis of ss invasion based on an irregular LHEL had the highest sensitivity and accuracy among the EUS imaging parameters (sensitivity 97.1% (33/34), specificity 86.7% (13/15), accuracy 93.8% (46/49)). When the deep group was limited to patients with a tumor depth of ss, the results were similar. When an irregular LHEL was used, the diagnostic accuracy of GBMTs with ss invasion was not significantly different between EUS specialists and beginners. Conclusions The observation of an irregular (thickened or narrow) LHEL observed on EUS could be a reliable and simple method of diagnosing GBMTs with ss invasion and could contribute to choosing an appropriate surgical method.