To reduce microvascular free flap failure (MFF), monitoring is crucial for the early detection of malperfusion and allows timely salvage. Therefore, the aim of this study was to evaluate hyperspectral imaging (HSI) in comparison to micro-doppler sonography (MDS) to monitor MFF perfusion in an in vivo rodent model. Bilateral groin flaps were raised on 20 Sprague–Dawley rats. The femoral artery was transected on the trial side and re-anastomosed. Flaps and anastomoses were assessed before, during, and after the period of ischemia every ten minutes for overall 60 min using HSI and MDS. The contralateral sides’ flaps served as controls. Tissue-oxygenation saturation (StO2), near-infrared perfusion index (NPI), hemoglobin (THI), and water distribution (TWI) were assessed by HSI, while blood flow was assessed by MDS. HSI correlates with the MDS signal in the case of sufficient and completely interrupted perfusion. HSI was able to validly and reproducibly detect tissue perfusion status using StO2 and NPI. After 40 min, flap perfusion decreased due to the general aggravation of hemodynamic circulatory situation, which resulted in a significant drop of StO2 (p < 0.005) and NPI (p < 0.005), whereas the Doppler signal remained unchanged. In accordance, HSI might be suitable to detect MFF general complications in an early stage and further decrease MFF failure rates, whereas MDS may only be used for direct complications at the anastomose site.