Because of the low GC content of the gene population, amino acids of the two mycoplasmas tend to be encoded by synonymous codons with an A or T end. Compared with the codon usage of ovine, Mycoplasma capricolum and M. agalactiae tend to select optimal codons, which are rare codons in ovine. Due to codon usage pattern caused by genes with key biological functions, the overall codon usage trends represent a certain evolutionary direction in the life cycle of the two mycoplasmas. The overall codon usage trends of a gene population of M. capricolum subsp. capricolum can be obviously separated from other mycoplasmas, and the overall codon usage trends of M. agalactiae are highly similar to those of M. bovis. These results partly indicate the independent evolution of the two mycoplasmas without the limits of the host cell’s environment. The GC and AT skews estimate nucleotide composition bias at different positions of nucleotide triplets and the protein consideration caused by the nucleotide composition bias at codon positions 1 and 2 largely take part in synonymous codon usage patterns of the two mycoplasmas. The correlation between the codon adaptation index and codon usage variation indicates that the effect of codon usage on gene expression in M. capricolum subsp. capricolum is opposite to that of M. agalactiae, further suggesting independence of the evolutionary process influencing the overall codon usage trends of gene populations of mycoplasmas.