Objective: To determine the diagnostic accuracy of optical coherence tomography (OCT) to assess surgical margins of canine soft tissue sarcoma (STS) and determine the influence of observer specialty and training. Study design: Blinded clinical prospective study. Animals: Twenty-five dogs undergoing surgical excision of STS. Methods: In vivo and ex vivo surgical margins were imaged with OCT after tumor resection. Representative images and videos were used to generate a training presentation and data sets. These were completed by 16 observers of four specialties (surgery, radiology, pathology, and OCT researchers). Images and videos from data sets were classified as cancerous or noncancerous. Results: The overall sensitivity and specificity were 88.2% and 92.8%, respectively, for in vivo tissues and 82.5% and 93.3%, respectively, for ex vivo