Abstract: CDPK are a multigene family, and play important roles in plant calcium signal transduction. The CDPKs that consist of Arabidopsis thaliana, N. tabacum and other species in tobacco genus were used to search the N. tomentosiformis genome databases. Then, the CDPK genes were isolated from N. tomentosiformis. The objectives of this investigation were to further study CDPK sequence characteristics, evolutionary relationship and gene expression. The results indicated that full-length genomic DNA sequences of 25 CDPK genes were obtained from N. tomentosiformis. Phylogenetic and principal coordinate analysis divided the 25 CDPKs into 4 subfamilies. The results of reverse transcription-PCR (RT-PCR) showed that the expression patterns of the 25 CDPK genes were significantly different. This study provides an important foundation for further functional dissection of the tobacco CDPK gene family.