Hypermethylation of CpGs in promoter regions and subsequent changes in gene expression are common features in acute myeloid leukemia (AML). Genome-wide studies of the methylome are not only useful to understand changes in DNA methylation and gene regulation but also to identify potential targets for antileukemic treatment. Here we performed methylated DNA immunoprecipitation (MeDIP) in the AML cell line HL-60 and donor-derived CD34+ cells, followed by hybridization on a human promoter tiling array. The comparative analysis of HL-60 versus CD34+ cells revealed differentially methylated promoter regions including genes that are frequently methylated in AML, such as p15/INK4B, OLIG2, RARß2 and estrogen receptor. Microarray data was validated by quantitative pyrosequencing. We corroborate previous reports that MeDIP, in our study combined with a promoter tiling array (MeDIP-Chip), is a robust method to identify genes that are differentially methylated in AML cells in a genome-wide manner, and is thus useful to identify new epigenetic targets for therapeutic or prognostic research.