Mastectomy skin flap necrosis (MSFN) and partial DIEP (deep inferior epigastric artery perforator) flap loss represent two frequently reported complications in immediate autologous breast reconstruction. These complications could be prevented when areas of insufficient tissue perfusion are detected intraoperatively. Hyperspectral imaging (HSI) is a relatively novel, non-invasive imaging technique, which could be used to objectively assess tissue perfusion through analysis of tissue oxygenation patterns (StO %), near-infrared (NIR%), tissue hemoglobin (THI%), and tissue water (TWI%) perfusion indices. This prospective clinical pilot study aimed to evaluate the efficacy of HSI for tissue perfusion assessment and to identify a cut-off value for flap necrosis. Ten patients with a mean age of 55.4 years underwent immediate unilateral autologous breast reconstruction. Prior, during and up to 72 h after surgery, a total of 19 hyperspectral images per patient were acquired. MSFN was observed in 3 out of 10 patients. No DIEP flap necrosis was observed. In all MSFN cases, an increased THI% and decreased StO %, NIR%, and TWI% were observed when compared to the vital group. StO % was found to be the most sensitive parameter to detect MSFN with a statistically significant lower mean StO % (51% in the vital group versus 32% in the necrosis group, < 0.0001) and a cut-off value of 36.29% for flap necrosis. HSI has the potential to accurately assess mastectomy skin flap perfusion and discriminate between vital and necrotic skin flap during the early postoperative period prior to clinical observation. Although the results should be confirmed in future studies, including DIEP flap necrosis specifically, these findings suggest that HSI can aid clinicians in postoperative mastectomy skin flap and DIEP flap monitoring.