Several developmentally important genomic regions map within the piebald deletion complex on distal mouse chromosome 14. We have combined computational gene prediction and comparative sequence analysis to characterize an approximately 4.3-Mb segment of the piebald region to identify candidate genes for the phenotypes presented by homozygous deletion mice. As a result we have ordered 13 deletion breakpoints, integrated the sequence with markers from a bacterial artificial chromosome (BAC) physical map, and identified 16 known or predicted genes and >1500 conserved sequence elements (CSEs) across the region. The candidate genes identified include Phr1 (formerly Pam) and Spry2, which are mouse homologs of genes required for development in Drosophila melanogaster. Gene content, order, and position are highly conserved between mouse chromosome 14 and the orthologous region of human chromosome 13. Our studies combining computational gene prediction with genetic and comparative genomic analyses provide insight regarding the functional composition and organization of this defined chromosomal region.