Athlete horses' contraction and conduction of the healthy heart influences racing performance. Gene expression patterns in the horse heart are not yet fully investigated. We aimed to evaluate the gene expression from the four chambers of the heart overall and with focus on genes involved in the electrophysiology of the heart in Thoroughbred racehorses with no clinical cardiac abnormalities. Tissue was collected from the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV). Total RNA was analysed by microarray technique. We compared gene expression in the heart chambers by contrasting atrial against ventricular chambers (chamber related differences), and by contrasting left side to right side (left-to-right related differences). The pathway analyses revealed that RA was characterised by significantly lower expression of genes related to energy derivation and metabolism in comparison to both ventricles and left atria. LA, on the other hand, was characterised by higher expression of genes related to cardiac conduction, and less expression of cardiac morphogenesis, compared to ventricles. The left-to-right related comparisons indicated wider differences between the atria than between the ventricles. Mapping of the genes specifically involved in cardiac conduction and contraction indicated clear chamber related differences. Some potassium channels, KCNE1 and KCNJ2,3 and 4, showed distinct atrial-ventricular specificity and genes involved in calcium regulation were, as a group, more abundant in both atria compared to ventricles. Our results provide a general overview of the gene expression pattern in the healthy racehorse, with particular focus on the cardiac ion channels.