Microorganisms, as is the case for all cells, must accurately reproduce their genomes and physically move newly replicated chromosomes into correct positions so that two genetically identical daughters are formed at cell division. New DNA synthesis in eubacteria, archaea, and eukaryotic microbes initiates from specialized DNA regions, termed replication origins. During every generation, staged assembly of origin DNA-initiator protein complexes controls loading of replicative helicases and recruits replication fork machinery. These initiator proteins are remarkably similar for all domains of life, but each microbe uses different strategies to control timing of initiation during the cell cycle and to ensure once and only once per-cycle initiation at any single origin. In eukaryotic microbes, movement of newly replicated chromosomes is performed by a microtubule-based mitotic spindle, but the prokaryotic chromosome segregation mechanism is less well understood. In eubacteria, ordered chromosomal structure is maintained and chromosome locations are correlated with specific cellular spaces throughout the cell cycle. This feat is accomplished by substitutes for the mitotic spindle, including motor proteins that rapidly separate replication origins and independently position all chromosomal loci as DNA replication precedes. These features suggest that prokaryotic segregation mechanisms are likely to be coupled to the DNA replication machinery.