Neural tube defects (NTDs), such as spina bifida, are among the most common serious birth defects, occurring in 0.5-10 per 1000 live births globally. Clinical studies have shown that periconceptional supplementation with folic acid (FA) can reduce NTD’s by up to 70%. In response to this data, the US government introduced FA fortification of enriched cereal grain products in 1996, and mandated it in 1998. The program has been associated with a decline of 20% in anencephaly and a decline of 34% in spina bifida. In despite of this, the mechanism(s) by which FA prevents NTDs is still not fully understood and is an area of active research. The Lrp6 (low-density lipoprotein-receptor related protein 6) gene provides two mice models of NTD that exhibit altered prevalence under periconceptional FA supplementation. Crooked tail mice (Lrp6Cd/+) contain a gain-of-function point mutation that results in exencephaly (development of the brain outside of the skull) in homozygotes, and are rescued by FA supplementation. Lrp6 deficient mice (Lrp6-/-) show a range of developmental defects, including spina bifida and exencephaly, however, FA supplementation increases their rates of absorption and embryonic lethality. We investigated the hypothesis that FA acts through changes in DNA methylation, by assaying the methylomes of heterozygous Cd and KO E9.5 mice with enhanced Reduced Representation Bisulfite Sequencing (eRRBS). To analyze this data, we created multiDiff, a new R package which extends the functionality of the Mason lab’s methylKit software for methylation analysis. multiDiff is designed to work with any number of binary covariates, allowing for the analysis and visualization of multiple terms and their interactions. It implements the novel maximum difference estimate for assigning biologically meaningful effects to coefficients, allowing for improved filtering of data. multiDiff displays consistently superior performance while varying simulation parameters as measured by AUC compared to DSS-general, a competing method (p<2.2-16, paired t-test). Integrative analyses was performed on DNA methylation and RNA-seq from embryonic mice on both Lrp6 backgrounds, Rn45s showed FA-associated differential expression and differential promoter methylation on the KO background. The analysis suggests that FA primarily acts in an independent rather than additive or combinatorial manner on methylation and expression and has wide ranging but mild effects throughout the epigenome. Genes associated with an independent mechanism of action were enriched for transcriptional regulation. On the Cd background, we noted genes affected by FA that had known links to Lrp6 biology, with the greatest number being associated with RhoA, suggesting involvement in the planar cell polarity Wnt signaling pathway. Methylation was also assayed in whole blood from P2 animals in order to identify biomarkers associated with Lrp6 mutation status and FA supplementation. Control cohorts were constructed across the time points to eliminate sites associated with tissue or developmental differences, as well as sex-specific versions of such differences. We found 43 persistently differentially methylated sites associated with the Cd mutation, and 25 with the KO mutation. Persistently differentially methylation loci associated with FA was identified at 86 sites in the Cd background, and 208 in the KO background. Before concluding, we discuss preliminary analyses of epigenetic and genetic data in human NTD patients, with a focus on ongoing challenges in the field. Tissue, sex, age and ancestry can all effect methylation levels, and must properly be accounted for in study design and analysis. In genetic analyses, correcting for ancestry requires large sample sizes to achieve the statistical power needed to confidently detect enrichment for multifactorial and polygenic disorder such as NTDs. The continuing growth of public datasets, especially in other neurological disorders such as autism, combined with advances in sequencing technology, and improvements in analytical methods are giving modern NTD researchers the tools to overcome these challenges.