Introduction: Constipation is one of the most common causes of referral to pediatric gastroenterology clinics. The histopathology and Immunohistochemistry (IHC) studies indicated that several neurons are currently detectable in motility of the gut. We aimed to evaluate calretinin in the children with chronic constipation. Materials and Methods In this cohort study, 40 children with chronic constipation, 28 boys (70%) and 12 girls (30%) referring to pediatric surgery ward in Shahid Mohammadi Hospital, Bandar Abbas-Iran, between January 2010 to February 2012, were recruited. Then, full rectal biopsy 1.5 cm above dentate line was performed for all children and calretinin immunoreactivity and pattern of staining for ganglion cells (nuclear and cytoplasmic) and also nerve fibers in different layers of the bowel (lamina propria, muscularis mucosa, submucosa, and muscularis propria) were measured. According to their pathologic results, the children were assigned in two groups for treatment: 1- Those with aganglionosis underwent pull- through operation, and 2- Patients with ganglion but abnormality in their immunohistochemistry received botulinum toxin. Results In this study, 9 (23.9%) AGANGLIONIC patients with the mean age of 3.6±1.7 years compared with 31 (76.1%) hypoganglionosis patients with the mean age of 3.2±1.2 years were examined. Pull- through operations were carried out for all patients in the AGANGLIONIC group, and for 6 (19.4%) patients in the hypoganglionosis group. Postoperative manometry was significantly better in both groups, but monomeric change was not significant between the two groups. Conclusion Our results revealed that calretinin is absent in AGANGLIONIC children, moreover we indicated, calretinin IHC is a very useful and valuable technique for detecting aganglionosis in patients with chronic constipation.