Introduction/objective Gastric conduit (GC) is used for reconstruction after esophagectomy. Anastomotic leakage (AL) incidence remains high, given the extensive disruption of the gastric circulation. Currently, there is no reliable method to intraoperatively quantify gastric perfusion. Hyperspectral imaging (HSI) has shown its potential to quantify serosal StO 2 . Confocal laser endomicroscopy (CLE) allows for automatic mucosal microcirculation quantification as functional capillary density area (FCD-A). The aim of this study was to quantify serosal and mucosal GC’s microperfusion using HSI and CLE. Local capillary lactate (LCL) served as biomarker. Methods GC was formed in 5 pigs and serosal StO 2 % was quantified at 3 regions of interest (ROI) using HSI: fundus (ROI-F), greater curvature (ROI-C), and pylorus (ROI-P). After intravenous injection of sodium-fluorescein (0.5 g), CLE-based mucosal microperfusion was assessed at the corresponding ROIs, and LCLs were quantified via a lactate analyzer. Results StO 2 and FCD-A at ROI-F (41 ± 10.6%, 3.3 ± 3.8, respectively) were significantly lower than ROI-C (68.2 ± 6.7%, p value: 0.005; 18.4 ± 7, p value: 0.01, respectively) and ROI-P (72 ± 10.4%, p value: 0.005; 15.7 ± 3.2 p value: 0.001). LCL value at ROI-F (9.6 ± 4.7 mmol/L) was significantly higher than at ROI-C (2.6 ± 1.2 mmol/L, p value: 0.04) and ROI-P (2.6 ± 1.3 mmol/L, p value: 0.04). No statistically significant difference was found in all metrics between ROI-C and ROI-P. StO 2 correlated with FCD-A (Pearson’s r  = 0.67). The LCL correlated negatively with both FCD-A (Spearman’s r  =  − 0.74) and StO 2 (Spearman’s r  =  −  0.54). Conclusions GC formation causes a drop in serosal and mucosal fundic perfusion. HSI and CLE correlate well and might become useful intraoperative tools.