In the recent years, the amount of wild-caught Procambarus clarkii has declined rapidly and it was under threat by white spot syndrome virus (WSSV). The absolute quantification of a virus in the host tissue is of great significance to understand the pathogenicity of the virus. However, quantitative analysis of WSSV in the tissues of infected P. clarkii was not reported. In this report, the epidemic of WSSV in red swamp crayfish from five cities in Hubei province was investigated. More than 80% of red swamp crayfish were WSSV positive when the DNA extracted from gills of red swamp crayfish were subjected to PCR assay. In addition, we also investigated the presence of WSSV antigen in the infected red swamp crayfish by Western blot using WSSV-VP28 protein specific serum generated in rabbit. The results showed that a specific band correlated with WSSV-VP28 was present in WSSV PCR-positive samples, but not in WSSV PCR-negative sample. To better quantify the amount of WSSV in the infected tissues, we established an absolute quantitative real-time PCR assay (qRT-PCR) for WSSV using recombinant plasmid pGEX-5x-1-VP28. Briefly, plasmid pGEX-5x-1-VP28 containing WSSV-VP28 gene was purified and quantified. Certain amount of pGEX-5x-1-VP28 was diluted serially and used as templates for qRT-PCR using primers specific for VP28 gene amplification. In doing so, a standard curve for the absolute quantification of WSSV was made. The amounts of WSSV genome that existed in the tissues were further measured by the qRT-PCR using the same above-mentioned primers specific for VP28 gene amplification. The absolute amounts of WSSV genomes in the tissues were obtained by comparison of the values of the qRT-PCR with those in the standard curve. Six tissues of red swamp crayfish, including gill, stomach, intestine, haemolymphocyte, hepatopancreas and heart, were sampled and used for WSSV quantification by the absolute qRT-PCR assay. The results showed that the highest most amounts of WSSV genome (about 10⁸ copies/mg) were detected in the gill, stomach and intestine, followed by haemolymphocyte (about 10⁷ copies/mg), hepatopancreas (about 10⁶ copies/mg). The lowest amount of WSSV was observed in the heart of infected red swamp crayfish, indicating that the replication of WSSV was tissue dependent. The high infection rate of WSSV in the crayfish cultured in the ponds (more than 80% of red swamp crayfish were WSSV positive) indicated that the cultured red swamp crayfish was persistently infected with WSSV.