Enterovirus 71 (EV71) is the major cause of hand-foot-and-mouth disease in children. In our study, using the complete genome sequences of 42 EV71 representing all three genotypes, we analyzed synonymous codon usage and the relative dinucleotide abundance in EV71 genome. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in EV71 genome. Furthermore, we observed that the relative abundance of dinucleotides in EV71 is independent of the overall base composition but is still the result of differential mutational pressure, which also shapes codon usage. In addition, other factors, such as hydrophobicity and aromaticity, also influence the codon usage variation among the genomes of EV71. This study represents the most comprehensive analysis of EV71 codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.