Eyespot is one of the most important fungal diseases of the stem base of wheat (Triticum aestivum L.). The presented study clearly demonstrated that the Pch1 gene was the main effective source for reducing the eyespot disease score in the analyzed winter wheat lines. Nevertheless, Pch1 was present only in 8−9% of the investigated lines. Using an isoenzymatic marker and molecular markers, the presence of the Pch1 gene and lack of the Pch2 gene was identified in six lines. Two lines, SMH 9409 and DL 358/13/4, were polymorphic in an isoenzymatic marker study. In the remaining three lines, C 3373/11-1, KBH 15.15 and KBP 1416, the Pch1 gene was identified only with the use of an isoenzymatic marker. Both genes Pch1 and Pch2, as well as the resistant variety Rendezvous, were found in three lines: DD 248/12, KBP 15.2 and STH 4431. In line DD 708/13, the presence of the Pch1 and Pch2 genes was identified, where the association between the Pch1 and the locus of the Xorw5 marker was broken. It was shown that the presence or absence of Pch1 and Pch2 genes did not significantly affect the grain yield (from the plot), although the yield was highest in the presence of both genes. A significant effect of the presence of the Pch1 gene on thousand kernel weight (TKW) was observed. Lines with the Pch1 gene showed significantly higher TKW values than lines without both genes or with the Pch2 gene only.