Ralstonia eutropha strain H16 is a facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium belonging to the family Burkholderiaceae of the Betaproteobacteria. The genome of R. eutropha H16 consists of two chromosomes (Chr1, Chr2) and one megaplasmid (pHG1), and thus shows a multi-replicon architecture, which is characteristic for all members of the Burkholderiaceae sequenced so far. The genes for housekeeping cell functions are located on Chr1. In contrast, many characteristic traits of R. eutropha H16 such as the ability to switch between alternative lifestyles and to utilize a broad variety of growth substrates are primarily encoded on the smaller replicons Chr2 and pHG1. The latter replicons also differ from Chr1 by carrying a repA-associated origin of replication typically found on plasmids. Relationships between the individual replicons from various Burkholderiaceae genomes were studied by multiple sequence alignments and whole-replicon protein comparisons. While strong conservation of gene content and order among the largest replicons indicate a common ancestor, the resemblance between the smaller replicons is considerably lower, suggesting a species-specific origin of Chr2. The megaplasmids, however, in most cases do not show any taxonomically related similarities. Based on the results of the comparative studies, a hypothesis for the evolution of the multi-replicon genomes of the Burkholderiaceae is proposed.