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The aim of this study was to develop an effective method for the identification of Vibrio harveyi based on using the toxR gene as a taxonomic marker.Primers for the toxR gene were designed for specificity to V. harveyi, and incorporated in a polymerase chain reaction (PCR). The results of the PCR, which took <5 h from DNA extraction to amplification, revealed positive amplification of the toxR gene fragment in 20 V. harveyi isolates including type strains, whereas DNA from 23 other Vibrionaceae type strains and 13 Vibrio parahaemolyticus strains were negative. The detection limit of the PCR was 4.0 × 103 cells ml−1. In addition, the technique enabled the recognition of V. harveyi from diseased fish.The PCR was specific and sensitive, enabling the identification of V. harveyi within 5 h.The PCR allowed the rapid and sensitive detection of V. harveyi.