Tuatara, Sphenodon punctatus, is the last survivor of the distinctive reptilian order Rhynchocephalia and is a species of extraordinary zoological interest, yet only recently have genomic analyses been undertaken. The karyotype consists of 28 macrochromosomes and 8 microchromosomes. A Bacterial Artificial Chromosome (BAC) library constructed for this species has allowed the first characterization of the tuatara genome. Sequence analysis of 11 fully sequenced BAC clones (approximately 0.03% coverage) increased the estimate of genome wide GC composition to 47.8%, the highest reported for any vertebrate. Our physical mapping data demonstrate discrete accumulation of repetitive elements in large blocks on some chromosomes, particularly the microchromosomes. We suggest that the large size of the genome (5.0 pg/haploid) is due to the accumulation of repetitive sequences. The microchromosomes of tuatara are rich in repetitive sequences, and the observation of one animal that lacked a microchromosome pair suggests that at least this microchromosome is unnecessary for survival. We used BACs bearing orthologues of known genes to construct a low-coverage cytogenetic map containing 21 markers. We identified a region on chromosome 4 of tuatara that shares homology with 7 Mb of chicken chromosome 2, and therefore the orthologous region of the snake Z chromosome. We identified a region on tuatara chromosome 3 that is orthologous to the chicken Z, and a region on chromosome 9 orthologous to the mammalian X. Since the tuatara determines sex by temperature and has no sex chromosomes, this implies that different tuatara autosome regions are homologous with the sex chromosomes of mammals, birds and snakes. We have identified anchor BAC clones that can be used to reliably mark chromosomes 3-7, 10 and 13, some of which are difficult to distinguish based on morphology alone. Fluorescence in situ hybridization mapping of 18S rDNA confirms the presence of a single NOR located on the long arm of chromosome 7, as previously identified by silver staining. Further work to construct a dense physical map will lead to a better understanding of the dynamics of genome evolution and organization in this isolated species.