Quantitative real-time polymerase chain reaction (qPCR) is a sensitive, efficient and reproducible technique for studying gene expression. Identification of stably expressed reference genes is required to avoid bias in these studies yet mostly unvalidated reference genes are used in studying gene expression in Clostridium difficile. Here, we sought to identify a set of stable reference genes used to normalize C. difficile expression data comparing exponential versus stationary phases of growth. Eight candidate reference genes ( rpoA, rrs, gyrA, gluD, adk, rpsJ, tpi, and rho) were assessed in 3 C. difficile genotypes (ribotypes 027, 078, and 001). The primers were analyzed for efficiency and the 8 genes were ranked according to their stability. Overall, the genes rrs, adk, and rpsJ ranked among the most stable. Identification of the most stable genes was, however, strain dependent and suggests that selection of reference genes in a heterogeneous species, such as C. difficile, requires multiple genes to be assessed to confirm their stability within the strains being studied.