Abstract: Two bands of OPAY02 type marker have been cloned, sequenced and two pair of primers was designed according to the two bands DNA sequence to amplify the SCAR-markers on 86 individuals randomly selected from New Yangzhou chickens. The results showed that the large molecular weight band is located on 3rd chromosome comparing with red jungle fowl genomic DNA, the sequence identities were 98% and 8 SNP mutations were detected , they were 195 (T→G), 316 (A→T), 538 (G→A), 731 (T→A), 1 147 (G→A), 1 329 (T→C), 1 927 (C→T) and 2 081 (C→T), the small molecular weight band can’t be found in red jungle fowl genomic DNA, speculated that New Yangzhou chicken may originate from not only red jungle fowl, but also other jungle fowls. It was also confirmed by SCAR-markers that the two bands of OPAY02 type marker can be applied to genetic analyses because of its stability and reliability. Genotype equilibrium test showed that New Yangzhou chickens are equilibrium at the two bands loci, two bands of OPAY02 type marker MAS will be in favor of breeding of chickens.