Abstract Pebrine is a most deadly disease of silkworm caused by infection with the microsporidium Nosema bombycis with extensive economic losses as a result of mortality in the end-stage of training. Several diagnosis methods have been established for detection of the disease, but most of these approaches are very old and rely on microscopic inspection and therefore are low in accuracy and speed. In the present study, a PCR-based method using a pair of specific primers only and a real-time PCR approach based on a specific probe were applied for detection of all pebrine-causing strains. Accuracy of the performance was investigated through DNA purification, PCR conditions, visualized PCR products and real-time PCR analysis. The results showed that present techniques had good ability to screen the suspicious samples with a high accuracy. The cost of providing a real-time PCR will be declined by increasing the sample sizes, and therefore, could be considered as a complementary or alternative method for pebrine inspection.