Description: The evolutionary factors in influencing the genetic characteristics of nucleotide, synonymous codon, and amino acid usage of 18 mycoplasma species were analyzed. The nucleotide usage at the 1st and 2nd codon position which determines amino acid composition of proteins has a significant correlation with the total nucleotide composition of gene population of these mycoplasma species, however, the nucleotide usage at the 3rd codon position which affects synonymous codon usage patterns has a slight correlation with either the total nucleotide composition or the nucleotide usage at the 1st and 2nd codon position. Other evolutionary factors join in the evolutionary process of mycoplasma apart from mutation pressure caused by nucleotide usage constraint based on the relationships between effective number of codons/codon adaptation index and nucleotide usage at the 3rd codon position. Although nucleotide usage of gene population in mycoplasma dominates in forming the overall codon usage trends, the relative abundance of codon with nucleotide context and amino acid usage pattern show that translation selection involved in translation accuracy and efficiency play an important role in synonymous codon usage patterns. In addition, synonymous codon usage patterns of gene population have a bigger power to represent genetic diversity among different species than amino acid usage. These results suggest that although the mycoplasmas reduce its genome size during the evolutionary process and shape the form, which is opposite to their hosts, of AT usages at high levels, this kind organism still depends on nucleotide usage at the 1st and 2nd codon positions to control syntheses of the requested proteins for surviving in their hosts and nucleotide usage at the 3rd codon position to develop genetic diversity of different mycoplasma species. This systemic analysis with 18 mycoplasma species may provide useful clues for further in vivo genetic studies on the related species.