Context: The main challenge in radical prostatectomy is complete excision of malignant tissue, while preserving continence and erectile function. Positive surgical margins (PSMs) occur in up to 38% of cases, are associated with tumour recurrences, and may result in debilitating additional therapies. Despite surgical developments for prostate cancer (PCa), no technology is yet implemented to assess surgical margins of the entire prostatic surface intraoperatively. Objective: The aim of this systematic review is to provide an overview of novel imaging methods developed for intraoperative margin assessment in PCa surgery, which are compared with standard postoperative histopathology. Evidence acquisition: A literature search of the last 10 yr was conducted in the Scopus, PubMed, and Embase (Ovid) databases. Eligible articles had to report the PSM rate according to their intraoperative margin assessment technology in comparison with standard histopathology. Evidence synthesis: The search resulted in 616 original articles, of which 11 were included for full-text review. The main technical developments in PCa margin assessment included optical coherence tomography, photodynamic diagnosis with 5-aminolevulinic acid, spectroscopy, and enhanced microscopy. These techniques are described and their main advantages, limitations, and applications in the clinical setting are discussed. Conclusions: Several imaging methods are suggested in literature for the detection of positive margins during PCa surgery. Despite promising qualifications of the mentioned technologies, many struggle to find implementation in the clinic. Surgical conditions hampering the signal, long imaging times, and comparison with histopathology are mutual challenges. The next step towards reduction of PSMs in PCa surgery includes evaluation of these technologies in large clinical trials. Patient summary: In this review, new technologies are reported that can assist the surgeon by detecting insufficient removal of all tumorous tissue during surgery, instead of the standard postoperative histopathological assessment. Currently, it is not clear whether these technologies improve the patient outcome directly; however, the review shows potential future implementations.