Abstract: In order to identify cultures of Alternaria Nees from China forestry culture collection center at molecular level by ITS sequence analysis. The internal transcribed spacer (ITS) region DNA fragment of 375 strains were amplified and sequenced by universal primers ITS1 and ITS4. And all the sequences of the 375 strains in present study were compared with the ITS sequences of related Alternaria species available in the GenBank database by BLAST search. Phylogenetic analysis was performed using the neighbor-joining method based on Paup 4.0 b10. ITS sequencing and NCBI BLAST results showed that: classification status of 21 strains from 375 trains were wrong, in which 9 strains had 92%-96% similarities with the reference strains, and the other 12 strains had 60%-85% similarities with the reference strains. The phylogenetic tree showed that 29 species were formed one cluster with 100% boot strap support, and the other seven species were divided into 6 clusters. The results indicated that: the ITS sequence could not be used to the separation of 29 species of Alternaria, but the other 6 species could be differentiated each other.