Three strains of Vibrios have been reported to cause shrimp disease, type strain of Vibrio harveyi and Vibrio campbellii, and a variant strain of Vibrio harveyi that was described to exhibit significant sequence difference of toxR and hemolysin genes from type strain V. harveyi. The early detection of these Vibrios causing shrimp disease is necessary to prevent disease outbreak. This study was focused on the use of three sets of PCR primers that target genes for ornithine decarboxylase, ToxR, and an unknown gene to detect and distinguish type strains of V. harveyi and V. campbellii and the variant V. harveyi that are pathogenic to black tiger shrimp (Penaeus monodon). Multiplex PCR profile revealed two bands (a 900-bp fragment of ornithine decarboxylase gene and a 175-bp fragment of an unknown gene) for type strain Vibrio harveyi (NBRC 15634), one band representing a 245-bp toxR gene fragment for type strain Vibrio campbellii (NBRC 15631), and two bands (a 900-bp odc gene fragment and a 245-bp toxR gene fragment) from isolate SW-9702, a representative of the variant V. harveyi. By using digoxigenin-labeled dUTPs in multiplex PCR, amplicons are labeled and could serve as probes for hybridization with blots containing known gene templates to confirm the amplification of target genes. The 900-bp labeled amplicons from type strain and variant V. harveyi hybridized with the V. harveyi odc gene blot. As expected, the labeled 175-bp amplicon of type strain V. harveyi hybridized with the blotted 175-bp putative marker of type strain V. harveyi, while the toxR amplicon from V. campbellii hybridized with blotted toxR of type strain /i>V. campbellii. Hybridization signal of the V. campbellii toxR blot with the multiplex PCR products from type strain V. harveyi was much reduced compared to the signal observed with multiplex PCR products from the variant strain of V. harveyi, because V. campbellii toxR exhibits only 76% sequence similarity with toxR of type strain V. harveyi and 92-93% similarity with the variant strain of V. harveyi. Results of multiplex PCR with reverse hybridization, as an indirect means to confirm the amplification of target genes, could distinguish the shrimp pathogenic strains of Vibrios used in the study.