Genetic diversity and maternal ancestry line relationships amongst a sample of 96 Cleveland Bay horses were investigated using a 479bp length of mitochondrial D-loop sequence. The analysis yielded at total of 11 haplotypes with 27 variable positions, all of which have been described in previous equine mitochondrial DNA d-loop studies. Four main haplotype clusters were present in the Cleveland Bay breed describing 89% of the total sample. This suggests that only four principal maternal ancestry lines exist in the present-day global Cleveland Bay population. Comparison of these sequences with other domestic horse haplotypes (Fig 2) shows a close association of the Cleveland Bay horse with Northern European (Clade C), Iberian (Clade A) and North African (Clade B) horse breeds. This indicates that the Cleveland Bay horse may not have evolved exclusively from the now extinct Chapman horse, as previous work as suggested. The Cleveland Bay horse remains one of only five domestic horse breeds classified as Critical on the Rare Breeds Survival Trust (UK) Watchlist and our results provide important information on the origins of this breed and represent a valuable tool for conservation purposes.