Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on the gyrB gene were carried out using a newly developed primer pair for gyrB. The 16S rRNA gene was also sequenced for comparison. A 1.2-kb fragment of the gyrB gene of 17 Gordonia strains including type strains was determined by direct sequencing of PCR amplified fragments. A total of 25 strains (8 of which were retrieved from a public database) of the genus Gordonia form a distinct phyletic line in the GyrB-based tree and are separated from other closely related species of genera of the suborder Corynebacterineae. Sequence similarity of the gyrB sequence from twelve Gordonia type strains ranged from 79.3 to 97.2%, corresponding to between 270 and 41 nucleotide differences, while there was only a 0.3-3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on the GyrB sequence deduced from the gyrB gene is consistent with that of DNA-DNA hybridization results and provides a better discrimination within the species of Gordonia compared to the 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia.