Résumé : Background The study investigates the diagnostic value of calretinin immunohistochemical staining (CIS) on rectal suction biopsies (RSB) in Hirschsprung's disease (HD). Methods A prospective study was conducted at Children's Hospital 2 in Ho Chi Minh City, Vietnam, from January through December 2015. Patients suspected of HD during this period underwent RSB and were followed in order to assess the accuracy of the diagnostic test with CIS compared with conventional histology (H&E). Results A total of 188 children with RSB were investigated. Median age was 7.1 (range 0.2–159) months with 65.4% boys. HD was confirmed in 80 (42.6%) children. There were 1 false positive and no false-negative cases. The sensitivity and specificity were 100% (80/80) and 99.1% (107/108) for CIS and 100% and 85.2% for H&E, respectively. Cohen's kappa coefficient was 0.9891 with a diagnostic accuracy of 99.5% for CIS, compared with 0.8303 and 91.5% for H&E, respectively. There were no serious complications related to the RSB. Conclusion RSB with CIS is a useful diagnostic method for HD, with easy interpretation and no need for cryostat. CIS has a high diagnostic accuracy and should be considered as the primary method for the diagnosis of HD by RSB. Level of evidence: Diagnostic Studies – Level I.