This chapter reviews the current knowledge on the organization and replication of bacterial genomes with a special emphasis on the initiation of DNA replication. Most bacteria contain one chromosome, however a few species, like the human pathogen Vibrio cholerae, carry a secondary chromosome. We discuss structures of origins of replication, the function of initiator proteins, the cell cycle regulation of initiation of replication, and the loading and assembly of replisomes. This is predominantly described for Escherichia coli, but parallels are drawn to Bacillus subtilis along with important pathogenic bacteria when relevant.