The 7.5k promoter of vaccinia virus Tiantan strain has been cloned by DNA polymerase chain reaction (PCR) method. Results of DNA sequencing analysis showed that in comparison with P7.5k of WR strain, three site mutations and 7 bp of natural deletion existed in the 155 bp fragment of P7.5k of Tiantan strain; four of 7 base pairs deleted were in the late transcriptional initiation region. Although the total mutation rate was high to 6.45%, these two 7.5k promoters of Tiantan strain & WR strain were all early-late promoters and not obviously different in their activities and functional phases. The results above confirmed further that it is a mechanism to keep their genetic stability that some genes of vaccinia virus have multiple transcriptional initiation sites and produce many mRNA with heterologous 5' ends.