Using a glnII (encoding glutamine synthetase II) PCR selective screening, a Frankia ACN14a gene library clone was isolated. A derived glnII-hybridising 2.7-kb HindIII subclone was characterised. Identities of 95% and 93% were observed, respectively, with the corresponding Frankia CpI1 glnI and glnII regions. A variable segment of the glnII region was selected, PCR amplified from various Frankia genomes, sequenced, and used to investigate phylogenetic relationships within the genus. glnII phylogenetic inferences are well-resolved and allowed us to deduce evolutionary trends among Frankia. Frankia radiation seems to begin with a diversification according to the ability or not to infect actinorhizal plants. The infective strains are divided into two clusters matching plant-colonising specificities.