Technical Abstract: Quantitative real-time RT-PCR provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant development. Partial clones of nine L. perenne housekeeping genes were obtained by RT-PCR using degenerate primers designed from the corresponding genes in closely related species. Primers for quantitative RT-PCR were designed based on partial clone sequences. The housekeeping genes were evaluated for their expression stability in different tissues at various stages of development. The analysis found that eEF-1a and eIF-4a were the most stable and ß-TUB was the least stable of the genes tested. Analysis by geNorm indicated that the four most stably expressed housekeeping genes (eEF-1a, eIF-4a, 25S rRNA and GAPDH) should be utilized when normalizing gene expression during plant developmental studies. For root crown tissues at different stages of development, eIF-4a and 25S rRNA were the most stably expressed of the housekeeping genes tested. In leaf tissues, eEF-1a and UBQ5 were the most stable of the housekeeping genes tested. We found that using two housekeeping genes as reference genes is sufficient during RT-PCR gene expression studies when analyzing either root crown or leaf tissues during different stages of development.