The E8 gene is related to ethylene biosynthesis in plants. To explore the effect of the expression pattern of the E8 gene on different E8 promoters, the molecular evolution of E8 promoters was investigated. A total of 16 E8 promoters were cloned from 16 accessions of seven tomato species, and were further analysed. The results from 19 E8 promoters including three previously cloned E8 promoters (X13437, DQ317599 and AF515784) showed that the size of the E8 promoters varied from 2101 bp (LA2150) to 2256 bp (LA2192); their sequences shared 69.9% homology and the average A/T content was 74.9%. Slide-window analysis divided E8 promoters into three regions – A, B and C – and the sequence identity in these regions was 72.5%, 41.2% and 70.8%, respectively. By searching the cis-elements of E8 promoters in the PLACE database, mutant nucleotides were found in some functional elements, and deletions or insertions were also found in regions responsible for ethylene biosysnthesis (–1702 to –1274) and the negative effect region (–1253 to –936). Our results indicate that the size of the functional region for ethylene biosynthesis in the E8 promoter could be shortened from 429 bp to 113 bp (–1612 to –1500). The results of molecular evolution analysis showed that the 19 E8 promoters could be classified into four clade groups, which is basically consistent with evolution of the tomato genome. Southern blot analysis results showed that the copy number of E8 promoters in tomato and some other wild species changed from 1 to 4. Taken together, our study provides important information for further elucidating the E8 gene expression pattern in tomato, analysing functional elements in the E8 promoter and reconstructing the potent E8 promoter.