The accuracy of quantitative real-time PCR (qRT-PCR) depends on the stability of the reference gene used for normalization. In heading Chinese cabbage (Brassica rapa L. ssp. pekinensis), the most stable reference genes for qRT-PCR during flower bud development have not been elucidated. In this study, the statistical software geNorm was used to test eight candidate reference genes during flower bud development in male sterile (Ms) and fertile (Mf) plants. The result revealed that the stability order was Tub/GAPDH > Cyp > EF1a > U34559 > BrTip41 > Apr > 18S rRNA, Tub and GAPDH were the most stable genes [average expression stability (M) 0.614], and the combined use of six reference genes [pairwise variation (V) 0.15] was suggested to be the optimal reference gene for qRT-PCR during flower bud development. Furthermore, the expressions of BcPME31 during flower bud development normalized with the combined use of six reference genes and with GAPDH or Tub alone were compared; the various results also suggested that selection of the optimal reference gene was necessary for gene expression analysis.