The large number of organisms and of genes sequenced at the present time permits now to study molecular evolution in such lower clades as genera, species, and subspecies. Here, we focus our attention on the genus <i>Mycobacterium</i>, in which we examined codon and aminoacid usage in 13 species, and in 12 subspecies for a total of 8,836,513 codons from 26,755 sequences. Within the genus <i>Mycobacterium</i>, frequencies of codon and aminoacid usage correlate between species and between subspecies. In the groups studied, aminoacid molecular weight and codon degeneracy influence correlations between frequencies, while GC content is the main factor influencing the effective number of codons. The coding GC, which is highly correlated with total genomic GC content, seems to be the main factor influencing present synonymous codon usage in the genus. In particular, the GC content at the 3rd base position seems to shape heavily the effective number of codons, giving indication that here mutational bias dominates over translational selection. Evolutionary trees based on codon and aminoacid usage are consistent with traditional phylogenies of species within the genus.