Nicotiana benthamiana is an important research tool, a vaccine biofactory and a model plant for the economically important crop family Solanaceae. It is an ancient allotetraploid arising from interspecific hybridization events about 5 million years ago. Intergenomic interactions between the subgenomes of polyploids are predicted to induce epigenetic modifications. Though a few epigenomic studies are currently available in polyploids, the genome-wide histone modifications and methylome of N. benthamiana have not yet been studied. To systematically analyse the epigenomic features in N. benthamiana, we profiled the genome-wide distribution of active (H3K4me2, H3K4me3, H3K27ac) and repressive (H3K27me3, H3K9me2) histone marks by chromatin immunoprecipitation-sequencing and the DNA methylome at a single base resolution by performing whole-genome bisulfite sequencing. In N. benthamiana genome, DNA methylation and H3K9me2 are mainly localized on the intra-and intergenic transposable elements. Other marks are involved with the active gene regulation thus, mostly localized on transcription start site, promoters, enhancers and gene body. A significant percentage of peaks of active gene regulatory marks are localized in the intergenic regions. This may indicate their possible involvement with cis gene regulatory elements for remote regulation of gene activity. A comparison between the two subgenomes showed the average distribution of active and repressive epigenetic marks and DNA methylation levels to be similar, but there are significant differences between some specific homeologous loci. High levels of active histone marks were observed at loci corresponding to the highly expressed homoeolog, whilst repressive marks such as H3K9me2 and DNA methylation were either low or absent in these regions. The results indicate that variation in epigenetic status can be associated with differential homoeolog expression, and may provide a guide for manipulating the expression of important genes in polyploid crops. Furthermore, these resources will speed up our understanding of the epigenetic basis of polyploid crops.