Diagnosis of very short-segment Hirschsprung's disease (vsHD) by rectal suction biopsy is challenging as its aganglionic zone (AZ) overlaps with physiologic hypoganglionic zone and calretinin-positive mucosal nerves may extend from the transition zone (TZ) into AZ. We studied whether an increasing trend/gradient of calretinin-positive mucosal nerves along the distance from AZ toward TZ aids in diagnosis of HD. In this study, 46 rectal suction biopsies from non-HD and HD, and 15 pull-through specimens from short-segment HD were evaluated by mucosal calretinin immunostain (CI) and image processing and analysis (IPA) to measure pixel count (PC, the percentage of calretinin stained pixels in the mucosa). Consecutive longitudinal sections of proximal AZ toward distal TZ in HD pull-through specimens were utilized as a vsHD surrogate model. First, we studied variability of mucosal CI in non-HD biopsies along the distance from dentate line. Second, we determined a cutoff point of mucosal CI by IPA that separated non-HD versus HD and applied this cutoff to longitudinal sections from proximal AZ to distal TZ segments in HD pull-through specimens. Third, we studied whether an increasing trend of mucosal CI was universally observed in HD pull-through. Our findings included a significant variability in PC along the biopsy distance in non-HD cases. Positive mucosal CI was found in proximal AZ in 6 (43%) of 14 HD pull-through, among which 1 case lacked submucosal nerve hypertrophy in the proximal AZ. All 14 HD pull-through cases showed an increasing trend/gradient of PC from AZ toward TZ. Based on our findings, the presence or absence of mucosal CI positivity and submucosal nerve hypertrophy may not reliably diagnose vsHD in rectal suction biopsy. While we acknowledge that the density of mucosal innervation in variable contexts and anatomical locations is unknown and yet to be explored, our study suggests that an increasing trend of positive mucosal CI from AZ toward TZ by IPA might prove to be a useful tool for the diagnosis of vsHD in the future.