Abstract: Objective To evaluate the power of gyrB gene sequence analysis in the identification of Nocardia species. Methods Forty one Nocardia strains were selected, the amplification of gyrB gene was conducted by polymerase chain reaction (PCR) and the purified products were directly sequenced for further analysis. In addition, 21gyrB gene sequences of Nocardia species were downloaded from GenBank and used in the analysis. DNAStar software was used to analyze these sequences and calculate the similarity of each pairs. A phylogenetic tree based on gyrB gene was constructed by Neighbor-joining and Maximum Likelihood method with Mega 6.06 software. Results In the phylogenetic tree based on gyrB gene sequence, Nocardia species were accurately divided into different clusters corresponding to each species, especially the close species. The intro species sequence similarity of Nocardia gyrB gene was from 94.2% to 100%; and the inter species similarity was from 79.2% to 97.6%, the average similarity was 86.9%. Conclusion The gyrB gene sequence analysis is a rapid, accurate and specific method, which could be used to identify Nocardia strains at species level. It is more effective to use gyrB gene to identify Nocardia species than 16S rDNA.