Aims: To differentiate Vibrio harveyi from closely related Vibrio species by toxR sequence analysis and design primers for the specific detection of the shellfish pathogen. Methods and Results: The partial toxR homologue from the shellfish pathogen V. harveyi was isolated by PCR using degenerate primers. The 578-bp toxR fragment from V. harveyi , that exhibited highest homology with partial toxR of V. parahaemolyticus (68%), is predicted to encode for a polypeptide with 192 amino acid residues. Alignment of the V. harveyi toxR nucleotide and deduced amino acid sequence with those from other Vibrio species revealed the presence of the fairly characteristic conserved transcription activation and transmembrane domain as well as the divergent membrane tether region that may be targeted for the development of species-specific oligonucleotide primers. Consequently, PCR primers that could amplify a 390-bp gene fragment in V. harveyi were designed by targeting portions of the V. harveyi toxR that display variability with toxR sequences from other Vibrio species. The 390-bp-amplicon was detected in all V. harveyi strains examined except in the nontarget bacteria and unexpectedly, in two shrimp-derived strains (VIB 391 and STD 3-101) from Thailand and Ecuador. Results show that strains exhibiting the 390-bp amplicon mostly belong to the same cluster based on previous amplified fragment length polymorphism data while strains which were previously unclustered or unclassified did not display the 390-bp PCR product. Conclusions: The toxR sequence variation could differentiate V. harveyi from closely related Vibrio species. A PCR protocol amplifying a 390-bp fragment of the V. harveyi toxR was established and could be useful in the specific and rapid detection of the species. Significance and Impact of the Study: The molecular approaches reported in this study could facilitate the early diagnosis and surveillance of luminous vibriosis in hatchery-reared fish and shellfish species through rapid identification and specific detection of causal agent.