Abstract: Based on the conserved fragment of plant chloroplast genes (Genbank No. DQ006133.1), a primer-probe set specific for Amaranthus spinosus (ASP-1) and a primer-probe set specific for Amaranthus spp. (ASP-2) were designed. Screened by to the 27 species including 9 target species, 7 closely-related species and 11 other non-target species，the two primer-probe sets show good specificity. According to the real-time PCR tests results, the optimum amplification temperatures for ASP-1 and ASP-2 are 62℃ and 60℃ separately. Under optimal conditions ASP-1 and ASP-2 showed high efficiency in the detection of target plants with the detection limits of 0.03 ng·μL-1 for A.spinosus and 0.01 ng·μL-1 for Amaranthus spp., respectively. It is the first time to establish a real-time PCR method for rapid identification of Amaranthus spinosus and Amaranthus spp., and as an auxiliary method of morphological identification, it can be implemented accurately and effectively.