Purpose: We aimed to determine the frequency of RET mosaicism in Hirschsprung disease (HSCR), test whether it has been underestimated, and to assess its contribution to HSCR risk. Methods: Targeted exome sequencing (n = 83) and RET single-gene screening (n = 69) were performed. Amplicon-based deep sequencing was applied on multiple tissue samples. TA cloning and sequencing were conducted for validation. Results: We identified eight de novo mutations in 152 patients (5.2%), of which six were pathogenic mosaic mutations. Two of these patients were somatic mosaics, with mutations detected in blood, colon, and saliva (mutant allele frequency: 35-44%). In addition, germ-line mosaicism was identified in four clinically unaffected subjects, each with an affected child, in multiple tissues (mutant allele frequency: 1-28%). Conclusion: Somatic mutations of the RET gene are underrecognized in HSCR. Molecular investigation of the parents of patients with seemingly sporadic mutations is essential to determine recurrence risk in these families. © 2017 American College of Medical Genetics and Genomics.