Two standard molecules were constructed by molecular clone technology of Tilletia indica Mitra and Tilletia walkeri Castl.. The standard molecule for Tilletia indica Mitra contained two DNA fragments, one about 2297 bp DNA sequence from mitochondrial DNA, the other ITS sequence 710 bp. The standard molecule for Tilletia walkeri Castl. contained two DNA fragments, one about 2.3 kb DNA sequence from mitochondrial DNA, the other ITS sequence 710 bp. In this research, the two standard molecules were detected, the results show good specificity, homogeneity and stability which can satisfy the need of molecular detection of Tilletia indica Mitra and Tilletia walkeri Castl..