Due to the degeneracy of genetic code, most amino acids are coded by more than one codon (synonymous codons). The synonymous codons are not used at equal frequencies both within and between organisms. Of the total 33 herpesvirus gB genes, approximately 9.1% of the total gB genes had low codon bias (ENC<35), 72.7% of the gB genes had high ENC values (ENC>50), indicating that these gB genes had random codon usage in herpesviruses. There might be no direct correlation between the codon usage bias and the host, which indicates that the tRNA abundance of the host was not the main factor inﬂuencing the codon usage bias. A plot of ENC vs. GC3 indicates that mutational bias may be a more important factor than tRNA abundance in determining codon usage bias of herpesvirus gB genes. Pearson correlation coefﬁcients between the ENC value and corresponding GC%, cumulative GC% in 2nd (GC2%) and 3rd codon position (GC3%) of each herpesvirus gB gene were -0.621 (p<0.01), -0.656 (p<0.01) and -0.712 (p<0.01), respectively, which implies that significant correlations existed between them. But no significant correlations existed between ENC and cumulative GC% in 1st codon position of each herpesviral gB gene. Furthermore, significant correlations also existed between GC% and GC3% of 33 herpesvirus gB genes (r=0.856, p<0.01). So it seems that, GC content, and particularly GC content at the 3rd base position, contributing greatly to the effective number of codons, indicating that the mutational bias dominates over translational selection. Further analysis on the relationship between gene length and ENC of 33 herpesvirus gB genes demonstrated that the two factors were not correlated. Signiﬁcant correlations were found between the gene expression levels assessed by CAI value and ENC (r = -0.424, p<0.05) and GC3 values (r = 0.644, p<0.01).