<span class="paragraphSection"><div class="boxTitle">Abstract</div>Nucleosome spacing patterns in the genome form a unique signature of a given cell, reflecting its chromatin organization and gene expression. Recently, studies of nucleosome spacing have expanded substantially due to the development of novel experimental tools and increased analysis of human samples. This has yielded thousands of high-resolution nucleosome maps across many species and cell types, as well as multiple human datasets that span across different ages and health conditions. With the rapid increase in nucleosome mapping data, their analysis and interpretation have become critically important. Indeed, several discrepancies in nucleosome spacing have been reported recently, using different experimental methods. However, when nucleosome spacing is consistently analysed, it can be linked to biologically important processes: (i) active genomic regions are characterized by shorter distances between nucleosomes in comparison to inactive regions; (ii) cancer cells tend to have shorter distances in comparison to normal cells of the same type; and (iii) ageing usually increases distances between nucleosomes. In many cases, the underlying molecular mechanisms remain to be clarified. Here, we provide a critical analysis of this field, focusing on nucleosome spacing in different types of genomic regions and cell types, as well as changes in cell differentiation, cancer, and ageing.</span>