The Sleeping Beauty transposon is a recently developed non-viral vector that can mediate insertion of transgenes into the mammalian genome. Foreign DNA elements that are introduced tend to invoke a host-defense mechanism resulting in epigenetic changes, such as DNA methylation, which may induce transcriptional inactivation of mammalian genes. To assess potential epigenetic modifications associated with Sleeping Beauty transposition, we investigated the DNA methylation pattern of transgenes inserted into the mouse genome as well as genomic regions flanking the insertion sites with bisulfite-mediated genomic sequencing. Transgenic mouse lines were created with two different Sleeping Beauty transposons carrying either the Agouti or eGFP transgene. Our results showed that DNA methylation in the keratin-14 promoter and Agouti transgene were negligible. In addition, two different genomic loci flanking the Agouti insertion site exhibited patterns of DNA methylation similar to wild-type mice. In contrast, high levels of DNA methylation were observed in the eGFP transgene and its ROSA26 promoter. These results indicate that transposition via Sleeping Beauty into the mouse genome may result in a significant level of de novo DNA methylation. This may depend on a number of different factors including the cargo DNA sequence, chromosomal context of the insertion site, and/or host genetic background.