The mitochondrial DNA (mtDNA) molecule, carrying genes encoding for respiratory chain enzymes, is a primary candidate for demonstrating associations between genotype and athletic performance in mammalian species. In humans, variation at seven protein encoding mitochondrial loci has been implicated in influencing fitness and performance characteristics. Although thoroughbred horses are selected for racing ability, there have not been any previous reported associations between genotypes and racecourse performance. The multi-factorial nature of the inheritance of racing ability is an obvious complicating factor. However, mitochondrial gene variation may represent a measurable component contributing to performance variability. Previous population studies of thoroughbreds have shown the existence of D-loop variation. Importantly, we have observed that there is also independent and extensive functional mitochondrial gene variation in the current thoroughbred racehorse population and that significant associations exist between mtDNA haplotype, as defined by functional genes, and aspects of racing performance.