A non-stop, single tube and semi-nested polymerase chain reaction (PCR) technique with simple procedure was developed for simultaneous detection and grading the level of white spot syndrome infection in penaeid shrimp, Penaeus monodon. In this PCR procedure, three sense primers and one antisense primer with uniform annealing temperature of 55 °C were used. These primers amplify three PCR products (500, 300 and 200 base pairs [bp]) depending upon the severity of infection. Quantities of WSSV-DNA at 10 pg and greater gave three PCR products of 500, 300, 200 bp. A moderate concentration of WSSV-DNA, around 100 fg, gave two products of 300 and 200 bp and a low concentration of 1 fg or more gave only one PCR product of 200 bp. This PCR technique was assessed for early detection of WSSV in shrimp. In time-course infectivity experiments conducted on shrimp with WSSV, one PCR product (200 bp) was seen in hemolymph, tail tissue and gill at 3 h post infection (p.i.); two PCR products (300 and 200 bp) were seen in tail tissue, hemolymph, heart tissue and gill at 18 h p.i. At 30 h p.i., three PCR products (500, 300, 200 bp) were seen in all the organs tested. The samples collected from control animals showed negative for WSSV. © Springer 2005.