The etiology of imperforateanus, a major phenotype of anorectal malformation (ARM), is still unknown and not a single gene has been reported to be associated with it. We studied a Korean family with six affected members with imperforateanusacrossthreegenerations by whole exome sequencing and identified a missensemutation in the EBF2 gene (c.215C > T; p.Ala72Val). This mutation is completely segregated with the disease phenotype in the family and is evolutionarily highly conserved among diverse vertebrates. Also, this mutation was predicted to be functionally damaging. These results support that missensemutation in the EBF2 c.215C > T (p.Ala72Val) is very likely to contribute to the pathogenesis of ARM in this family.