Red rot disease, caused by the oomycete agent Pythium porphyrae, severely impacts the production of the alga Neopyropia (Pyropia). However, little is known about the pathogenetic mechanisms involved because of a lack of a genetic transformation system for this pathogen. Understanding the gene expression patterns of pathogens in response to different conditions is vital; however, this requires suitable reference genes for quantitative real-time polymerase chain reaction data normalization. In this study, 11 reference genes of P. porphyrae were evaluated at different developmental stages (vegetative growth, sporangia formation, zoospore formation, and zoospore release), temperatures (4, 15, 25, and 37 °C), salinities (0, 20, and 35), pHs (4, 7.5, and 10) and infection stages [5, 7, 9 days post infection (dpi)]. Various mathematical algorithms (ΔΔ-CT comparison method, BestKeeper, NormFinder, and geNorm) were used to evaluate the stability of the reference gene expression. Of the 11 genes, those encoding γ-Tubulin and α-Tubulin2 showed the best stability across the different developmental stages, Actin2 and 18S under different temperature conditions, GAPDH and EF1α under different salinity conditions, GAPDH and EIF2α under different pH conditions, and GAPDH and α-tubulin2 across different infection stages. Under all the tested conditions, GAPDH showed the best stability, indicating a reliable candidate as a universal reference gene for qRT-PCR analysis. To our knowledge, this is the first study to evaluate reference genes in P. porphyrae, with the aim of finding suitable candidate genes for the standardization of gene expression studies in this algal oomycete pathogen, and potentially other related pathogens. [ABSTRACT FROM AUTHOR]