Background and aims Colorectal cancer (CRC) is a major killer. Host immunity is important in tumorigenesis. Direct comparison among IL-36 alpha, IL-36 beta and IL-36 gamma in the prognosis of CRC is unclear. Methods CRC tissue arrays were generated from colorectostomy samples with TNM stage, invasion depth and the demography of these patients (n = 185). Using immunohistochemistry/histopathology, IL-36 alpha, IL-36 beta and IL-36 gamma were determined, in comparison to non-cancer tissues. Results A significant association was observed between colonic IL-36 alpha, IL-36 beta or IL-36 gamma and the presence of cancer (with all P < 0.0001). Using ROC curve analysis, specificity and sensitivity of IL-36 alpha, IL-36 beta or IL-36 gamma were confirmed, with area under the curve (AUC) values of 0.68, 0.73 and 0.65, respectively. Significant differences in survival were observed between IL-36 alpha(high) and IL-36 alpha(low) (P = 0.003) or IL-36 gamma(high) and IL-36 gamma(low) (P = 0.03). Survival curves varied significantly when further stratification into sub-groups, on the basis of combined levels of expression of two isotypes of IL-36 was undertaken. A significant difference was observed when levels of IL-36 alpha and IL-36 beta were combined (P = 0.01), or a combination of IL-36 alpha plus IL-36 gamma (P = 0.002). The sub-groups with a combination of IL-36 alpha(high) plus IL-36 beta(high), or IL-36 alpha(high) plus IL-36 gamma(low) exhibited the longest survival time among CRC patients. In contrast, the sub-groups of IL-36 alpha(low) plus IL-36 beta(high) or IL-36 alpha(low) plus IL-36 gamma(high) had the shortest overall survival. Using the log-rank test, IL-36 alpha(high) expression significantly improved survival in patients with an invasion depth of T4 (P < 0.0001), lymph node metastasis (P = 0.04), TNM III-IV (P = 0.03) or with a right-sided colon tumour (P = 0.02). Similarly, IL-36 gamma(low) expression was significantly associated with improved survival in patients with no lymph node metastasis (P = 0.008), TNM I-II (P = 0.03) or with a left-sided colon tumour (P = 0.05). Multivariate analysis demonstrated that among IL-36 alpha, IL-36 beta and IL-36 gamma, only IL-36 alpha (HR, 0.37; 95% CI, 0.16-0.87; P = 0.02) was an independent factor in survival, using Cox proportional hazards regression analysis. Conclusion IL-36 alpha or IL-36 gamma are reliable biomarkers in predicting the prognosis of CRC during the later or early stages of the disease, respectively. Combining IL-36 alpha plus IL-36 gamma appears to more accurately predict the postoperative prognosis of CRC patients. Our data may be useful in the management of CRC.