All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the differences in chromatin landscape of this unique collection will shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. N.benthamiana is also an important biotechnological tool and it is widely used in virological research and functional genomics. Chromatin Immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is well established technique used to study histone modifications. However, due to the starchy nature of mature N.benthamiana leaves, previously published protocols were unsuitable. The aim of this study was to optimise ChIP protocol for N.benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes.