A novel real-time PCR detection and quantification method for the red seabream iridovirus (RSIV) was developed using the major capsid protein (MCP) gene. A linear relationship was observed between the amount of input DNA and the threshold cycle (CT) values over a range of 1 to 109 viral copies. The real-time PCR was 2 orders of magnitude more sensitive than the conventional PCR. It was also shown to be specific to RSIV, i.e., it did not react with either hirame rhabdovirus (HIRRV), viral hemorrhagic septicemia virus (VHSV) or a DNA sample from apparently healthy red seabream Pagrus major. The viral load of dead juvenile red seabream (1 g average body weight) experimentally challenged with RSIV was in the range of 2.4 × 105 - 5.4 × 107 MCP gene copy number. To determine the target tissues of RSIV in the infected fish, 2 red seabream with average body weight of 50 g were experimentally challenged with RSIV and DNA was extracted from the various tissues and organs at 3rd day post-infection. Highest viral load was obtained in the heart with a mean MCP gene copy number of 2.7 × 105, while the blood had the lowest viral load at 8.8 × 103 mean MCP gene copy number. After the viral challenge, the real-time PCR assay was still able to detect RSIV from vaccinated survivors, which otherwise tested negative for RSIV by conventional PCR.