Frankia are nitrogen-fixing actinomycetes that form a symbiotic association with over 200 species of woody dicotyledonous plants. Recently, three Frankia genomes were completely sequenced. In this study, the synonymous codon usage patterns of three Frankia genomes (strains CcI3, ACN14a, and EAN1pec) were determined and compared to each other and to other actinobacteria. As expected for a high G+C organism, codon usage by Frankia was highly biased, but differences were observed among the three strains. Using the codon adaptation index (CAI) as a numerical estimator of gene expression level, highly expressed genes in Frankia were predicted with ribosomal protein genes as a reference. The analysis of the predicted highly expressed genes showed that Frankia strain CcI3 had a different profile from the other two strains. Strain CcI3 had fewer predicted highly expressed genes in several COG categories including lipid transport and metabolism, secondary metabolites biosynthesis, inorganic ion transport and metabolism, and general function prediction only than Frankia strains EAN1pec and ACN14a. Interestingly, Frankia EAN1pec had more predicted highly expressed genes in transcription and signal transduction mechanisms than the other two strains. These differences were not just a reflection in total gene numbers, but also based on percentage of genes within a category. These results support the hypothesis that strain CcI3 is becoming a symbiotic specialist and the other two facultative symbiotic strains are maintaining their capacity to exist as free-living soil dwellers.