Abstract: The screening of the most suitable internal reference genes for the study on the regulation of the expression of Hyphomicrobium has laid a foundation for greatly increasing the yield of Pyrroloquinoline Quinone (PQQ). The Real-time quantitative PCR technology was used to analyze the relative expression of 6 candidate reference genes in the genome of Hyphomicrobium. And the softwares of Best-keeper, geNorm and NormFinder were used to evaluate the expression stability of candidate reference genes in each growth period and determine the most appropriate reference genes. The results showed that through the analysis and evaluation on the transcription situation of the six candidate reference genes (16S rRNA、gapdh、recA、ldh、rpoB and S5) at four different growth periods of Hyphomicrobium, the six candidate reference genes all showed a greater stability and specificity. And according to the comprehensive assessment of the three softwares, it was determined that the two internal reference genes gapdh and recA could be expressed stably in the two strains at different periods. Therefore, the genes gapdh and recA with stable expression were selected from the six candidate reference genes for further study on gene expression regulation of Hyphomicrobium.