Since blood cells produce various soluble factors like cytokines or chemokines, gene expression analysis in whole blood could be important to investigate disease pathogenesis. In gene expression analysis with quantitative real-time RT-PCR, accurate determination of relative mRNA transcription levels requires appropriate reference genes. To identify the optimal reference gene in canine whole blood, we compared transcription levels of twelve candidate reference genes in total RNA extracted using the PAXgene system. The stability of the reference gene was evaluated by three different statistical programs, GeNorm, Normfinder and Bestkeeper. The results indicated that SDHA, CG14980 and TBP were the most stably expressed genes, which can be used as optimal reference genes for gene expression analysis in canine whole blood.