Purpose: In this prospective cross secitonal study, we aimed to investigate the adequacy, sensivity and specifity of rectal aspiration biopsy samples as the standart diagnosis of patients suspected HD for the histopathological diagnosis Material and methods: The study was performed between november 2016 and march 2018. We prospectively included 24 patients aged between 0-3 years that suspicious with HD to the study. After Rectosigmoid index (RSI) were calculated according to the contrast enhanced colon graphy, Patients who RSI<1 were performed rectal aspiration biopsy with the suspicious of HD. All patients’ clinical features, treatment options, complications, laboratory results and radiological findings were recorded for further analyses. Biopsy specimens were evaluated by the specialist patholog and findings were recorded. Results: There were no ganglion on the specimens of rectal aspiration biopsy in 10(%41.6) patients and diagnosed as HD. Ganglion cell was detected in 5(%20.8) patients and diagnosis of HD was excluded. Inadequate or suspicious biopsy specimens for histopathological evaluation was observed in 9(%37.5) patients. If the biopsy volume was greater than 4 mm3, sensitivity was 80% and specificity was 66.67% in diagnosis of HH (AUC = 0.789); If the Submucosa / Mucosa ratio is greater than 0.75 or the submucosa is greater than 0.42, the sensitivity and the specificity were 86.67% and 66.67%, respectively. Conclusion: In this prospective cross-sectional study, we demonstrated that rectal aspiration biopsy in the diagnosis of HD is easily feasible, safe, has higher sensitivity and specificity, and lower complication rates.