BACKGROUND: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. RESULTS: In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. CONCLUSIONS: Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements.