R391 and SXT are members of a group of eleven chromosome-borne conjugative elements found in the gamma-proteobacteria, whose members carry different antibiotic resistance traits. Recent genomic analysis of R391 and SXT revealed a highly conserved 'backbone' encoding integration/excision, conjugative transfer, and regulation functions, augmented by an array of phenotypic traits and transposable elements. In this study, PCR amplification and sequence analysis were employed to investigate the genomic structure of two further MGE of the R391 family, pMERPH (HgR) and R997 (ApR, SmR, SuR). R997 and pMERPH were found to be structurally related to R391 and SXT and share a number of virtually identical regions with them-including putative integration, conjugative transfer, and regulatory determinants-interrupted by variable DNA segments and transposable elements. The presence of a highly conserved backbone in the four elements strongly suggests their origin in a common ancestral element, which itself was a mosaic of sequences related to phages and plasmids. Subsequent genetic recombination and the acquisition of transposable elements resulted in the possession of variable phenotypic traits among the four MGE, and diversification into two distinct lineages, the first one including R391 and pMERPH, the second one containing SXT and R997.