The filamentous fungus Fusarium graminearum is an important cereal pathogen. Although quantitative realtime PCR (qRT-PCR) is commonly used to analyze the expression of important fungal genes, no detailed validation of reference genes for the normalization of qRT-PCR data has been performed in this fungus. Here, we evaluated 15 candidate genes as references, including those previously described as housekeeping genes and those selected from the whole transcriptome sequencing data. By a combination of three statistical algorithms (BestKeeper, geNorm, and NormFinder), the variation in the expression of these genes was assessed under different culture conditions that favored mycelial growth, sexual development, and trichothecene mycotoxin production. When favoring mycelial growth, GzFLO and GzUBH expression were most stable in complete medium. Both EF1A and GzRPS16 expression were relatively stable under all conditions on carrot agar, including mycelial growth and the subsequent perithecial induction stage. These two genes were also most stable during trichothecene production. For the combined data set, GzUBH and EF1A were selected as the most stable. Thus, these genes are suitable reference genes for accurate normalization of qRT-PCR data for gene expression analyses of F. graminearum and other related fungi.