Nucleotide sequences of approximately 213 bp of the nif H-D intergene and the beginning of nifD were determined for symbiotic Frankia isolates from the major host-infectivity groups. This region of the nif operon is variable enough to classify most infective Frankia strains at the species level. Phylogenetic inferences from these sequences are in agreement with the 16S rRNA-derived phylogeny of the genus and, thus, are in favor of an intrageneric evolution of nif genes by orthology. Phylogenetic lineages derived from combined nifH-D intergene and partial nifD and 16S rRNA sequences are supported for at least 93% of bootstrap replicates and are useful for investigating evolutionary relationships of the genus and symbiotic properties of this microorganism. The genus Frankia is divided into two major phylogenetic clusters that match with the separation of species according to the mechanism of infection of actinorhizal plants. One cluster groups species strictly adapted to the mechanism of root hair infection (RHI), and the other groups species adapted to the mechanism of direct intercellular penetration. In the RHI cluster, the species infective on Casuarina plants appears to have emerged from strains infective on Alnus. The concordance between the symbiotic properties and the molecular phylogeny of Frankia strains indicates a major role for the host plant in the evolution and speciation of the genus Frankia.