The human body consists of many trillions of cells harboring nearly identical genomes. Yet cells manifest strikingly different cell morphologies and functions, reflecting their distinct patterns of gene expression. One of the most fundamental questions in human biology is how one genome sequence can give rise to so many different cell types. Increasing evidence indicates that the spatial, three-dimensional (3D) organization of chromatin influences gene expression and cell fate. The spatial organization of metazoan genomes has a direct influence on fundamental nuclear processes that include transcription, replication, and DNA repair. Advancements in high-throughput genomics and computational methods in the past 15 years have taken our understanding of the genome to a whole new level by allowing genome-wide assessments of chromatin conformation in the 3D space. General principles guiding the spatial conformation of chromosomes, such as compartmentalization and formation of topologically associating domains and chromatin loops, are now becoming increasingly understood, and this is leading to a better understanding of long-range chromosomal communication. In this book, recent studies concerning the chromatin compaction, local interactions, long-range interactions and the nuclear positioning of each chromatin type are reviewed. The well-established and emerging technologies that are revolutionizing our understanding of higher-order genome architecture are summarized.