Little is known about the origin and evolution of supernumerary (B) chromosomes. This study utilizes molecular markers to examine the evolutionary history and microstructural organization of the supernumerary paternal-sex-ratio (PSR) chromosome of the parasitic wasp Nasonia vitripennis. Copies of the retrotransposon NATE were previously isolated from PSR and the genomes of N. vitripennis and related wasp species. A phylogenetic analysis of sequences representing 29 elements from PSR and seven wasp species, coupled with a hybridization analysis of elements in genomic DNA provides evidence that PSR was recently transferred into N. vitripennis from a species in the genus Trichomalopsis. A linear region of the PSR chromosome was compared by Southern blot analysis with genomic DNA from N. vitripennis, Nasonia longicornis, Trichomalopsis americanus, and Trichomalopsis dubius. A region organized similarly to the region on PSR was not evident in any of the species, thus a progenitor region was not identified. However, the hybridizations revealed that this region of PSR is primarily composed of repetitive sequences that appear dispersed in these wasp genomes, and might represent additional mobile elements. At least three different dispersed repeats are present in the 18 kb region of PSR. The abundance of tandem and dispersed repetitive sequences in this relatively small region provides additional evidence for the degenerate structure of the PSR chromosome.