The activities of DNA methyltransferases are important for a variety of cellular functions in bacteria. In this study, we developed a modified high-throughput technique called methyl homopolymer tail mediated sequencing (methyl HTM-seq) to identify the undermethylated sites in the Vibrio cholerae genome for the two DNA methyltransferases, Dam, an adenine methyltransferase, and VchM, a cytosine methyltransferase, during growth in rich medium in vitro. Many of the undermethylated sites occurred in intergenic regions, and for most of these sites, we identified the transcription factors responsible for undermethylation. This confirmed the presence of previously hypothesized DNA-protein interactions for these transcription factors and provided insight into the biological state of these cells during growth in vitro. DNA adenine methylation has previously been shown to mediate heritable epigenetic switches in gene regulation. However, none of the undermethylated Dam sites tested showed evidence of regulation by this mechanism. This study is the first to identify undermethylated adenines and cytosines genomewide in a bacterium using second-generation sequencing technology.