Hirschsprung's disease (HSCR) is a congenital disorder in which ganglion cells are absent in variable portions of the lower digestive tract according to which patients are classified. The RET gene is the major HSCR gene, although reduced penetrance of RET mutations and variable expression of HSCR phenotype indicates that more than one gene is required. An unidentified RET-dependent modifier on 3p21 appears to be necessary for transmission of the short HSCR (S-HSCR) phenotype. We investigated 6 Mb of the 3p21 region on a quest for the HSCR-susceptibility locus. Fifty-eight S-HSCR case-parent trios were genotyped using Sequenom technology for 214 tag single nucleotide polymorphisms (SNPs) distributed along 6 Mb of the 3p21 region. A five-marker haplotype, spanning a 118 kb gene-rich region, was found to be overtransmitted to affected offspring. The associated haplotype encompasses three genes involved in neurological phenotypes. Importantly, this association was replicated in an independent sample of 172 S-HSCR cases and 153 unrelated controls. Ranking markers by proximity to candidate genes or by expected functional consequences could be used in follow-up studies to finally pinpoint this HSCR locus.