Mitochondrial DNA of five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3 kb- EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, T. barclayana. The clone was partially sequenced, primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3 kb fragment from total DNA of 17 T. indica isolates from India, Pakistan and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, T. foetida, T. caries, T. fusca and T. controversa) did not produce any bands as detected by ethidium bromide-stained agarose gels and Southern hybridizations. Sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.