In analysis of DNA viruses from the contaminated shellfish using PCR, preparation method of template DNA is an important factor to get enough copy number of viruses. In this study, we evaluated the efficiency of PCR template of Megalocytivirus (sT50mg-D) DNA obtained from 50 mg digestive gland homogenate of oyster using commercial method, and compared with that obtained from 5 g of the same tissues (T5g-D) after PEG precipitation procedures of virus. Both templates DNA suspended in the same volume of distilled water showed positive results by primary PCR with 35 cycles, and the presence of Megalocytivirus was confirmed in oysters collected from cultured farms in Korea. Moreover, PCR with sT50mg-D allowed us to discriminate the contaminated oyster individually, that can not be done in PCR with T5g-D prepared from the mixture of three different individual oyster to get 5 g digestive gland homogenate. In quantitative analysis with real time PCR, Megalocytivirus concentrations in 50 templates prepared using 0.5~50 mg of one positive sample were appeared in the range 6.14E+00~1.2E+02/. We were not able to get positive result using template DNA contained less than 6.14E+00 copies. Consequently, 2-step PCR performed with DNA extracts from oyster homogenate of small amount (sT50mg-D) i) was enough to detect the contaminated Megalocytivirus in shellfish, ii) allowed us to do the analysis for individual shellfish rather than mixture of several shellfish and iii) showed the presence of Megalocytivirus in oyster from Korea.