Rapid and accurate identification of pathogen is a major quarantine strategy for outbreak prevention. We used capillary electrophoresis-random amplified polymorphic DNA (CE-RAPD) to generate highly discriminatory pathogen profiles, reduced batch effects between profiles by novel normalization procedure and pattern of technical repeats, followed by target similarity evaluation using target identification score (TIS). A full target signature contains several patterns. TIS system was optimized by training set isolates that included three species, and validated using two hundred clinical Klebsiella pneumoniae isolates. Hierarchical clustering analysis showed CE-RAPD profiles arrange clusters according to the species or the source. Moreover, samples with similar profile may display similar antibiotic susceptibility. By using a signature of four patterns, the TIS system could accurately identify target among different isolates. The variation between isolates may be caused by small change in genome. TIS system provides a standardized tool for building of outbreak firewall and facilitate data exchange.