Eyespot can reduce yields, even up to 50%. There are four genetically characterized resistances in wheat varieties, controlled by: (1) the Pch1 gene, transferred from Aegilops ventricosa; (2) the Pch2 gene, originating from wheat variety Capelle Desprez; (3) the Pch3 gene, originating from Dasypyrum villosum; and (4) the Q.Pch.jic-5A gene, a quantitative trait locus (QTL) located on chromosome 5A of Capelle Desprez. However, those loci have drawbacks, such as linkage of Pch1 with deleterious traits and limited effectiveness of Pch2 against the disease. Here we present an initial study which aims to characterize wheat pre-registration breeding lines carrying 12 eyespot resistance genes, consider their resistance expression in inoculation tests and the influence of resistance genotypes on the yield. We selected four groups of breeding lines, carrying: (1) the Pch1 gene alone: one line; (2) the Pch2 gene alone: four lines; (3) the Q.Pch.jic-5A gene alone: one line; and (4) Pch1 + Q.Pch.jic-5A: three lines. For the first time, the effect of the combination of Pch1 and Q.Pch.jic-5A genes was compared with resistance conferred by Pch1 or Q.Pch.jic-5A alone. We found significant differences between infection scores evaluated in resistant lines carrying Pch1 and Q.Pch.jic-5A alone, while no differences in terms of the level of resistance expression were detected between Pch1 alone and Pch1 + Q.Pch.jic-5A, and between wheat lines carrying Pch1 and Pch2 alone. Moreover, we demonstrated that the Pch1 gene, together with an Ae. ventricosa segment, caused statistically significant yield losses, both as a single eyespot resistance source or in a combination with Q.Pch.jic-5A. Yield scores showed that wheat lines with Q.Pch.jic-5A had the highest yields, similar to the yielding potential of Pch2-bearing lines and control varieties.