Background: Perioperative hyperglycemia is a known risk factor for postoperative complications after colorectal surgery. The aim of this study was to investigate whether intraoperative blood glucose values are associated with colorectal anastomotic leakage in diabetic and non-diabetic patients undergoing colorectal surgery. Methods: This is an additional analysis of a previously published prospective, observational cohort study (the LekCheck study). Fourteen hospitals in Europe and Australia collected perioperative data. Consecutive adult patients undergoing colorectal surgery with primary anastomosis between 2016 and 2018 were included. From all patients, preoperative diabetic status was known and intraoperative blood glucose was determined just prior to the creation of the anastomosis. The primary outcome was the occurrence of anastomotic leakage within 30 days postoperatively. Results: Of 1474 patients (mean age 68 years), 224 patients (15%) had diabetes mellitus, 737 patients (50%) had intraoperative hyperglycemia (≥126 mg/dL, ≥7.0 mmol/L), and 129 patients (8.8%) developed anastomotic leakage. Patients with intraoperative hyperglycemia had higher anastomotic leakage rates compared to patients with a normal blood glucose level (12% versus 5%, P<0.001). Anastomotic leakage rate did not significantly differ between diabetic and non-diabetic patients (12% versus 8%, P=0.058). Logistic regression analyses showed that higher blood glucose levels were associated with an increasing leakage risk in non-diabetic patients only. Conclusion: Incidence and severity of intraoperative hyperglycemia are associated with anastomotic leakage in non-diabetic patients. Whether hyperglycemia is an epiphenomenon, a marker for other risk factors or a potential modifiable risk factor per se for anastomotic leakage requires future research.