Nucleosomes are the basic unit of the three-dimensional structure of chromatin. It is now widely accepted that the positioning and occupancy of nucleosomes play important roles in fundamental genomic processes such as DNA transcription, replication and repair. Among the methods used to provide genome-wide nucleosomal positions and occupancy levels, MNase-seq has proven to be highly effective. Indeed, with this method, the nucleosomal landscapes of a variety of organisms have now been investigated, revealing both commonalities and differences. In this review, we first introduce the technical principles underlying MNase-seq, focusing on details essential to precisely resolve nucleosome positioning and occupancy. We then describe recent advances with this method, as well as future perspectives of its role in chromatin biology, with a particular focus of uncovering mechanistic insights of many disease process.