White spot syndrome baculovirus (WSBV) has been found across different shrimp species and in different Asian countries. The detection of WSBV in shrimp with white spot syndrome has already been achieved by means of 1-step polymerase chain reaction (PCR). In an attempt to establish a more sensitive assay, we evaluated the effect of 2-step amplification with nested primers on the sensitivity of WSBV diagnostic PCR. The sensitivity of the 2-step amplification was 10 super(3) to 10 super(4) times higher than that of 1-step amplification. Using both techniques, we successfully detected WSBV DNA in cultured and captured shrimp, crabs and other arthropods. Cultured Penaeus monodon (black tiger shrimp), P. japonicus (kuruma shrimp), P. penicillatus (red tail shrimp), and Metapenaeus ensis (sand shrimp) displaying white spot syndrome were collected from farms at different localities. One-step amplification of the DNA extracted from these shrimps consistently yielded an expected 1447 bp PCR product. Some of the tested specimens of cultured Scylla serrata (mud crab) that exhibited white spot syndrome were positive in 1-step WSBV diagnostic PCR, while others were positive only in 2-step WSBV diagnostic PCR. Use of the 2-step amplification protocol also detected a WSBV-specific DNA fragment in Macrobrachium rosenbergii (the giant freshwater prawn) exhibiting white spot syndrome. We also confirmed that WSBV exists in wild-caught shrimp (P. monodon, P. japonicus, P. semisulcatus and P. penicillatus) and crabs (Charybdis feriatus, Portunus pelagicus and P. sanguinolentus) collected from the natural environment in coastal waters around southern Taiwan. Detection of WSBV in non-cultured arthropods collected from WSBV-affected shrimp farms revealed that copepods, the pest crab Helice tridens, small pest Palaemonidae prawn and the larvae of an Ephydridae insect were reservoir hosts of WSBV. The relatedness between WSBV and Thailand's systemic ectodermal and mesodermal baculovirus (SEMBV) is discussed in this paper.