Motivation Despite conservation in general architecture of promoters and protein–DNA interaction interface of RNA polymerases among various prokaryotes, identification of promoter regions in the whole genome sequences remains a daunting challenge. The available tools for promoter prediction do not seem to address the problem satisfactorily, apparently because the biochemical nature of promoter signals is yet to be understood fully. Using 28 structural and 3 energetic parameters, we found that prokaryotic promoter regions have a unique structural and energy state, quite distinct from that of coding regions and the information for this signature state is in-built in their sequences. We developed a novel promoter prediction tool from these 31 parameters using various statistical techniques. Results Here, we introduce SEProm, a novel tool that is developed by studying and utilizing the in-built structural and energy information of DNA sequences, which is applicable to all prokaryotes including archaea. Compared to five most recent, diverged and current best available tools, SEProm performs much better, predicting promoters with an ‘F-value’ of 82.04 and ‘Precision’ of 81.08. The next best ‘F-value’ was obtained with PromPredict (72.14) followed by BProm (68.37). On the basis of ‘Precision’ value, the next best ‘Precision’ was observed for Pepper (75.39) followed by PromPredict (72.01). SEProm maintained the lead even when comparison was done on two test organisms (not involved in training for SEProm). Availability and implementation The software is freely available with easy to follow instructions (www.scfbio-iitd.res.in/software/TSS_Predict.jsp). Supplementary information Supplementary data are available at Bioinformatics online.