BACKGROUND: Hirschsprung’s disease (HD) is the major cause of pediatric intestinal obstruction. It occurs due to failure of migration of ganglion cells from neural crest, leads to development of aganglionic segments which varies from ultra short to total intestinal involvement. AIMS & OBJECTIVES: The aim of the study was to describe the characteristics of Calretinin immunostaining pattern in both ganglionic and aganglionic segments of colon in patients with Hirschsprung’s disease and to evaluate the role of Calretinin immunohistochemistry in the diagnosis of Hirschsprung’s disease. MATERIALS & METHODS: A prospective study was done in 80 cases. Patients with suspected and follow–up cases of Hirschsprung’s disease were taken who came for definitive surgery. Hematoxylin and eosin (H and E) staining was done for detection of ganglion cells followed by Immunohistochemical staining for Calretinin. RESULTS: During histopathological examination of the spastic segment by H&E staining, out of total 80 cases, Ganglion cells were seen in 14(17.5%) cases, absent in 50(62.5%) cases and 16 (20%) cases were suspicious for Ganglion cells. Calretinin immunoexpression was negative in all cases of aganglionic segments. 2(2.5%) cases that were reported as positive for Ganglion cells in H&E staining, were reported as negative by Calretinin IHC. So Calretinin had sensitivity of 96% and specificity of 100% in our study. The positive predictive value was 100% and negative predictive value was 92.59%. CONCLUSION: Calretinin IHC is accurate in detecting ganglion cells. It is extremely useful in solving the doubtful cases of HD. It can serve as a valuable and cost‑effective diagnostic aid in Hirschsprung’s disease.