Two fragments SCS362o and SCS3580 of the co-dominant marker OPS0362o&580 that were linked to the resistance gene of soybean frog-eye leaf spot have been completely sequenced. A significant insertion of 30 bp is the main reason of the polymorphism between the two fragments. The results of Southern hybridization indicate that SCS362o derives from a single- or low-copy sequence and can be used as an RFLP probe. A co-dominant SCAR marker SCSSeao&sao has been developed based on the sequences. The segregation of SCS362o&58o is similar to that of RAPD marker OPS03620&580. Significant polymorphism has been shown between resistant and susceptible genotypes when 62 soybean genotypes were surveyed for the SCAR marker. Therefore, the marker can be used in the resistance breeding of soybean frog-eye leaf spot by marker-assisted selection. Two fragments SCS362o and SCS3580 of the co-dominant marker OPS0362o & 580 that were linked to the resistance gene of soybean frog-eye leaf spot have been completely sequenced. A significant insertion of 30 bp is the main reason of the polymorphism between the two fragments. results of Southern hybridization indicate that SCS362o derives from a single- or low-copy sequence and can be used as an RFLP probe. A co-dominant SCAR marker SCSSeao & sao has been developed based on the sequences. The segregation of SCS362o & 58o is similar to that of RAPD marker OPS03620 & 580. Significant polymorphism has been shown to be resistant to susceptible genotypes when 62 soybean genotypes were surveyed for the SCAR marker. Thus, the marker can be used in the resistance breeding of soybean frog-eye leaf spot by marker-assisted selection.