Quantitative real-time polymerase chain reaction (qPCR) can be used to investigate the relative expression of selected genes under different conditions; hence, it requires a robust endogenous reference consisting of various constitutive genes, also known as housekeeping genes. Thorough scrutiny of several genes under the specific conditions of the later experiments provides the most stably expressed candidate genes. The pairwise comparison and variance analysis of these candidate genes also yield the minimum number of reference genes for reliable RT-qPCR analysis. Therefore, this study selected three reference genes for the normalization and relative quantification of the gene expression of a Komagataella phaffii (previously classified as Pichia pastoris) wild-type strain using dextrose and methanol as different carbon sources.