Purpose <p>Hirschsprung disease (HD) is defined by absent ganglion cells in the rectum and, often, by hypertrophic nerve fibers. However, these hypertrophic nerves may be lacking when aganglionosis extends beyond the sigmoid colon. This study examines histopathological differences in rectal biopsies from HD patients, focusing on hypertrophic nerves and their correlation with aganglionosis length. </p> Methods <p>This retrospective study analyzed patients with HD at a single institution from January 2016 to February 2025. Rectal biopsy and pull-through specimen pathology reports were reviewed to assess for hypertrophic nerves, focusing on differences among various lengths of HD.</p> Results <p>There were 256 patients included, with diagnosed short-segment (<i>n</i> = 209), long-segment (<i>n</i> = 19), total colonic (<i>n</i> = 13), small intestinal (<i>n</i> = 10), and total intestinal HD (<i>n</i> = 5). Hypertrophic nerves were absent on rectal biopsy in 13 of 209 short-segment and 15 of 47 patients with extended aganglionosis. Absence of hypertrophic nerves on rectal biopsy was predictive of aganglionosis beyond the sigmoid (OR = 6.13, 95% CI 2.73, 13.78), with sensitivity of 32.6% and specificity of 93.7%.</p> Conclusion <p>The absence of hypertrophic nerves suggests a more proximal extent of aganglionosis; however, their presence has poor sensitivity for ruling out long-segment variants. These findings could guide pre-operative counseling and surgical planning, though prospective evaluation is needed. </p>