Abstract: Onion, Welsh Onion, and their F1 progenies were applified to establish a method for interspecific hybrids identification by using PCR-RFLP. The results showed that rDNA internal transcription spacers (ITS) of onion could be digested into two restriction fragments with endonuclease Dde I ,while no Dde I restriction site was found in Welsh onion. Welsh onion hold a restriction fragment of approximately 0.3 kb after the PCR products of V7 region of mitochondria srRNA was digested with Rsa I , which was slightly shorter than that of onion. Of the 25 F1 hybrids analyzed in this paper, 22 were true F1, while the other three ones were false hybrids. The present research established a easy and accurate method for interspecific hybrids identification between onion and Welsh onion.