Abstract: A novel cDNA encoding the second subunit of NADH dehydrogenase (NDHB) was cloned from Fraxinus velutina Torr by RT-PCR and designated as FvndhB. The cDNA was 893 bp in length, encoding 288 amino acid with a predicted molecular mass of 32.4kD and a pI of 10.17. Sequence alignment showed that FvndhB presented higher similarity with other ndhBs at the level of nucleic acid. The similarity was the highest between Fraxinus velutina Torr and Morus alba, the second was Populus alba, and Cryptomeria Japonica was the last. Phylogeny tree was constructed by the software of DNAMAN, the genetic relationship was far between Fraxinus velutina Torr and other plants. In this research, FvndhB gene segment was obtained, and the phylogenetic analysis was researched. It established basis on cloning the span of FvndhB and studying the characteristics of FvndhB gene expression in Fraxinus velutina Torr.