The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.