Locating replication origins (ROs) is an old problem to which diverse answers have been previously given. However, a better and more convenient method still remains desirable because previously reported methods are time-consuming. In this study, we report an easier method for locating ROs based on real-time polymerase chain reaction. The feasibility of this method was demonstrated using Escherichia coli K-12 and Halobacterium salinarium NRC-1 as models. Our results show that this method is easy, fast, and accurate in locating ROs.