Abstract: White spot syndrome virus (WSS V) and Infectious hypoder mal and hae matopoietic necrosis virus (I HHNV) are responsible for significant econom ic loss in the shrm i p industry1 In order to sm i ultaneously and massively identifyWSSV and I HHNV, two pairs ofprm i ers and two Taq Man probeswere designed and synthesized according to the conserved gene sequences ofWSSV (AF3690-9) and I HHNV (AF-18--6) in GenBank1The reaction parameters such as the concentra-tion oftwo pair ofprm i ers, twoTaq Man probes and the reaction bufferwere optm i ized to develop amultiplex real-tm i ePCR assay f or the rapid detection ofWSS V and I HHNV1Themultiplex real-tm i e PCR assaywas found to be specific and be able to detect and d ifferentiateWSSV and I HHNV, and no positive resu lts were observed when nucleic acid fro m Vibrio, Taura Syndro meVirus and Streptococcuswereused asmultiplex real-tm iePCR templates1The developed multiplex real-tm i ePCR assaywas compared with that ofroutinePCR1The sensitivity ofmultiplex real-tm i ePCR assaywas-and 20 template cop-ies forWSS V and I HHNV respectively, and its sensitivity was 10 3and 10-tm i es h igher than that of the routine PCR1The sa mpleswere examined using themultiplex real-tm i ePCR repeatedly and the results indicated that themultiplex real-tm i e PCR was reproducible1Different concentrations ofWSS V and I HHNV could be identified when mixed together, which miplied the assay could be applied to clin ical confir mation for smiultaneous infection ofWSSV and I HHNV1Themultiplex re-al-tmie PCR results of 30 routine PCR positive sa mples showed that one specific amplified curve was displayed when shrm i p was infected by only one of these two viral pathogens, whereas t wo specific amplified curves were displayed when shrm i p was infected by two viral pathogens1The resu lt ind icated thatmultiplex real-tmie PCR was able to detect and d iffer-entiate the presence ofeach pathogen in infected clinical shrm i p1Thismultiplex real-tm i ePCR assay is a quick, sensitive, specific and quantitative tool f or detection ofWSSV and I HHNV, and itwill be useful for the controlofWSS and I HHN in shrmip.