As a scientist, one’s perspective of the human genome is informed by the way it is studied – at the level of single nucleotides, a single gene, a specific genomic region, an entire chromosome, the complete karyotype, or the nucleus that encompasses both the genome and the nuclear components that support genome structure, function, stability, and inheritance. Experimentally investigating aspects of genome structure and chromosome number and higher order packaging requires different technical approaches that offer varying levels of resolution. This special issue of Chromosome Research provides overviews of a few current methodologies to study chromosome and genome organization and function, with a particular focus on contemporary sequencing-based approaches.