Objective Elevated levels of plasma trimethylamine N-oxide (TMAO), the product of gut microbiome and hepatic-mediated metabolism of dietary choline and l-carnitine, have recently been identified as a novel risk factor for the development of atherosclerosis in mice and humans. The goal of this study was to identify the genetic factors associated with plasma TMAO levels. Approach and Results We used comparative genome-wide association study approaches to discover loci for plasma TMAO levels in mice and humans. A genome-wide association study in the hybrid mouse diversity panel identified a locus for TMAO levels on chromosome 3 (P=2.37x10(-6)) that colocalized with a highly significant (P=1.07x10(-20)) cis-expression quantitative trait locus for solute carrier family 30 member 7. This zinc transporter could thus represent 1 positional candidate gene responsible for the association signal at this locus in mice. A genome-wide association study for plasma TMAO levels in 1973 humans identified 2 loci with suggestive evidence of association (P=3.0x10(-7)) on chromosomes 1q23.3 and 2p12. However, genotyping of the lead variants at these loci in 1892 additional subjects failed to replicate their association with plasma TMAO levels. Conclusions The results of these limited observational studies indicate that, at least in humans, genes play a marginal role in determining TMAO levels and that any genetic effects are relatively weak and complex. Variation in diet or the repertoire of gut microbiota may be more important determinants of plasma TMAO levels in mice and humans, which should be investigated in future studies.