Pch1 gene translocated from Aegilops ventricosa provides effective resistance to eyespot in wheat. To track the Pch1 gene introgression, we investigated 372 genotypes obtained from various breeding programs using endopeptidase EpD1b marker, sequence-tagged-site (STS) marker XustSSR2001-7DL, and the score of infection index (K-index) evaluated after in vivo inoculation test. These genotypes were divided into three groups with 136, 124 and 112 genotypes for the field test lasting three years. In 2011, the mean K-index was 0.81, while 2012 and 2013 the mean K-indexes were 1.60 and 1.46, respectively. Both marker results indicated that 18 genotypes possessed Pch1 gene. Statistical analysis of the level of K-index showed that these 18 genotypes were resistant to eyespot, which verified the proper assignment of wheat genotypes with Pch1 gene based on the marker data. Thus, the endopeptidase and XustSSR2001-7DL are useful for identifying sources of eyespot resistance gene Pch1 in wheat breeding program.