PURPOSES: The preoperative diagnosis of Hirschsprung disease (HD) requires a rectal mucosal biopsy and acetylcholinesterase (AchE) staining. However, the optimal timing for performing these procedures remains unclear. In this study, we assessed the most effective timing for a rectal biopsy and AchE staining to diagnose HD. METHODS: We retrospectively collected data from 57 patients who underwent radical surgery for HD at Mie University Hospital between January 2008 and April 2024. We reviewed all rectal biopsies, including those with multiple samples, and analyzed the biopsy date and the results of AchE staining (positive or negative). RESULTS: Among the 57 children with a confirmed HD diagnosis, 36 were included in the study. Fifty-two rectal biopsy sessions were performed in these 36 patients with HD, including 15 with multiple biopsies. A receiver operating characteristic analysis showed that the optimal age for diagnosing HD was older than 21 days. Biopsies performed at ≤ 3 weeks of age had a low diagnostic accuracy rate of 30.8%, whereas rectal biopsies performed at >
3 weeks of age showed a high positive diagnostic rate of 87.5%. CONCLUSIONS: Rectal mucosal biopsies performed after 3 weeks of age show a higher diagnostic accuracy for HD. CLINICAL TRIAL NUMBER: Not applicable.