Abstract We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. We found that differentially methylated regions are enriched within CpG islands. While non-CG sites with low methylation levels are not conserved, heavily methylated non-CG sites are strongly conserved especially when found within the motif TACAG. By combining BS-seq and RNA-seq data we identified 1080 genes that are significantly allelically imbalanced. Intermediately methylated CG sites are found within a significant fraction of allelically imbalanced genes as well, suggesting that these sites may be regulating allele specific transcription. Finally, we identified variations in methylation levels across the three lines at transcription factor binding sites and found several transcription factors that showed a correlation between methylation and gene expression levels, suggesting that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.