The discovery of modifier genes in human and mouse has seen slow progress. In mice, this is largely due to the reliance on inherent variability among inbred strains, which makes identifying the causal variant, even in highly resolved mapped loci, quite challenging. We have developed a fast and cost-effective method for genetic mapping of causal ENU-induced mutations using Next Generation Sequencing that combines single nucleotide polymorphism (SNP) discovery, mutation localization, and identification of causal sequence variants. This approach precludes the need for an outcross to facilitate mapping, as we use the ENU-induced variants as SNP markers. This strategy allows us to perform both primary screens and modifier screens on fully inbred lines. We have successfully mapped causal mutations using this approach, and we are currently testing the efficacy of this strategy with regard to modifier discovery using two C57BL/6 congenic strains as our sensitized lines. One is a loss-of-function allele of the SHH transcriptional effector Gli2, which can be modified by prenatal ethanol exposure to generate holoprosencephaly-like phenotypes. The other is an ENU-induced loss-of-function allele of Pibf1 that causes cleft lip/palate and ciliopathy-like phenotypes. Both alleles could reveal modifiers of genes involved in craniofacial, brain, and skeletal development as well as those involved in the formation and function of the primary cilium. In the simplest cases, whole-genome sequencing will reveal areas of homozygosity shared between mutants with a consistent “modified” phenotype, and allow us to narrow our focus to the induced variants included in these homozygous regions. We anticipate that we may ascertain more complex genetic interactions as well as new ENU-induced mutant phenotypes that model human birth defects. In our first screen we discovered a mutation in Colgalt1(collagen beta (1-O) galactosyltransferase type 1), which is required for proper galactosylation of hydroxylysine residues in a number of collagens. Mutant embryos exhibit skeletal and muscular defects, and we hypothesize that the Colgalt1 mutant could serve as a model of a human connective tissue disorder and/or congenital muscular dystrophy or myopathy. The mutants also show variable expressivity of cleft palate and exencephaly. Examination of the sequencing data appears to exclude the likelihood that these are due to an unlinked ENU-induced variant, illustrating that stochastic effects remain an important source of variation in developmental phenotypes. Funding: NIH R01 HD36404.