Partial rpoD, rpoB, and 16S rRNA gene sequences were obtained from databases and (or) amplified from 12 strains of Frankia. These strains belonged to either Cluster 1 (Alnus-, Myrica-, Comptonia-, and Casuarina-infective strains) or Cluster 3 (Elaeagnus-infective strain). An rpoD gene-based PCR approach was designed to allow the detection of frankiae in complex samples. Additionally, partial gene sequences obtained using 2 rpoB gene primer sets (named rpoB-1 and rpoB-2) were used to generate phylogenetic eurograms to find a molecular tool able to assess biodiversity among Frankia strains. The rpoB-2 primer set allowed separation of closely related strains and groupings representative of host plant compatibility groups. One exception to this was for strains ACN10a and ACN14a, isolated from the same geographical location. Results obtained showed that rpoB-2 is a tool of great interest to evaluate relatedness of Frankia strains, and assess biodiversity in this genus. Additionally, since rpoB-2 phylogenetic profiles of the Frankia strains studied reflected the species of host plants they were isolated from, the study of rpoB (a house-keeping gene) shows promise for future ecological studies on these symbioses.