Intraoperative assessment of colonic viability can be challenging and largely subjective. Objective methods are often impractical. Viability is related to re-establishment of colonic perfusion; particularly microvascular perfusion. This study evaluated the utility of dark-field microscopy (DFM) of the colonic serosa as an objective method of assessing microperfusion. To measure microvascular perfusion indices (MPI) of the pelvic flexure serosa in horses with surgical colonic lesions and correlate these with macroperfusion indices (MaPI) and histomorphometry. Prospective, clinical, case-control study. Control horses and horses with colonic volvulus (LCV), displacement, and/or simple obstruction undergoing surgery had DFM video loops performed on the pelvic flexure. Total vessel density, perfused vessel density, proportion of perfusion vessels and microvascular flow index were calculated from video analysis. Macroperfusion indices (arterial blood pressure and heart rate) were recorded. Histomorphometry was used to determine a mucosal injury score. Differences between lesions for MPI, MaPI and histomorphometry were compared using ANOVA or Kruskal-Wallis statistic. Spearman correlations between MPI with MaPI were performed. Linear regression was used to assess the relationship between MPI and histomorphometry. P<0.05 was significant. Horses with LCV had lower perfused vessel density, proportion of microvascular perfusion vessels and flow index than horses with nonstrangulating obstructions and control horses. Macroperfusion indices were not correlated with MPI but MPI were correlated with histomorphometry. Dark-field microscopy is achievable in the operating room and can quantify MPI from the colonic serosa in different colonic lesions. Macroperfusion indices were not related with colonic MPI. Microvascular perfusion indices can predict the severity of histopathological change at the pelvic flexure. Derangements of MPI may be more useful indicators of colonic pathology and viability and offer a more objective assessment of intestinal injury than subjective methods. Further study is needed to determine the utility of DFM in predicting survival in horses with LCV.