Abstract Background Regulation of transcription by DNA methylation in 5’-CpG-3’ context is a widespread mechanism allowing differential expression of genetically identical cells to persist throughout development. Consequently, differences in DNA methylation can reinforce variation in gene expression among cells, tissues, populations, and species. Despite a surge in studies on DNA methylation, we know little about the importance of DNA methylation in population differentiation and speciation. Here we investigate the regulatory and evolutionary impact of DNA methylation in five tissues of two Ficedula flycatcher species and their naturally occurring F 1 hybrids. Results We show that the density of CpG in the promoters of genes determines the strength of the association between DNA methylation and gene expression. The impact of DNA methylation on gene expression varies among tissues with the brain showing unique patterns. Differentially expressed genes between parental species are predicted by genetic and methylation differentiation in CpG-rich promoters. However, both these factors fail to predict hybrid misexpression suggesting that promoter mismethylation is not a main determinant of hybrid misexpression in Ficedula flycatchers. Using allele-specific methylation estimates in hybrids, we also determine the genome-wide contribution of cis- and trans effects in DNA methylation differentiation. These distinct mechanisms are roughly balanced in all tissues except the brain, where trans differences predominate. Conclusions Overall, this study provides insight on the regulatory and evolutionary impact of DNA methylation in songbirds.