Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.