IntroductionWe previously developed an artificial intelligence (AI) model for automatic coronary angiography (CAG) segmentation, using deep learning. To validate this approach, the model was applied to a new dataset and results are reported.MethodsRetrospective selection of patients undergoing CAG and percutaneous coronary intervention or invasive physiology assessment over a one month period from four centers. A single frame was selected from images containing a lesion with a 50–99% stenosis (visual estimation). Automatic Quantitative Coronary Analysis (QCA) was performed with a validated software. Images were then segmented by the AI model. Lesion diameters, area overlap [based on true positive (TP) and true negative (TN) pixels] and a global segmentation score (GSS – 0 -100 points) - previously developed and published - were measured.Results123 regions of interest from 117 images across 90 patients were included. There were no significant differences between lesion diameter, percentage diameter stenosis and distal border diameter between the original/segmented images. There was a statistically significant albeit minor difference [0,19 mm (0,09–0,28)] regarding proximal border diameter. Overlap accuracy ((TP + TN)/(TP + TN + FP + FN)), sensitivity (TP / (TP + FN)) and Dice Score (2TP / (2TP + FN + FP)) between original/segmented images was 99,9%, 95,1% and 94,8%, respectively. The GSS was 92 (87–96), similar to the previously obtained value in the training dataset.Conclusionthe AI model was capable of accurate CAG segmentation across multiple performance metrics, when applied to a multicentric validation dataset. This paves the way for future research on its clinical uses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10554-023-02839-5.