Genomes of 144 human DNA viruses were analyzed in the aspect of their compositional asymmetry. DNA viruses were divided into two groups according to their genome sizes. The analysis revealed that the level of guanine and cytosine (GC content) in the coding sequences of small genome DNA viruses was significantly lower than that of large genome DNA viruses. Because small genome viruses replicate their genomes using cellular enzymes, while large genome viruses use their own enzymes for genome replication, the two groups of viruses may be under different mutational bias and/or selection pressure. In these viruses, GC content at the third codon position correlated with GC content at the first and second codon position. However, the relationship in small genome DNA viruses was weaker than that in large genome DNA viruses, suggesting that their genome composition may be more strongly influenced by codon usage preference or restriction on amino acid composition.