Intra-operative surgical margin assessment by pathology is labor-intensive and time-consuming and is not practically capable of sampling the entire specimen. Positive surgical margins (PSMs), or tumor extending to the surface of the excised specimen, are associated with increased tumor recurrence and are accepted as poor independent prognostic indicators. Considering the PSM rate is high for patients with prostate and kidney cancer, residual tumor following radical prostatectomy and partial nephrectomy remains a significant problem. To address the unmet clinical need for an imaging tool that can provide sub-cellular resolution images of large areas of excised surgical specimens in an intra-operative timeframe, we have developed a video rate structured illumination microscopy (VR-SIM) system. We conducted a clinical trial using VR-SIM to create gigapixel mosaics of entire margin surfaces for each specimen. In the ongoing study, 5 patients undergoing radical prostatectomy and 4 patients undergoing partial nephrectomy participated to have digital images of their surgical specimens reviewed in comparison to the pathology report. The surfaces of the intact, excised specimens were imaged in an appropriate timeframe and showed visualization of histopathologically relevant structures.