The quantity and quality of protein and the rheological traits of wheat are crucial for processing flour in the baking industry, but there are few comparisons in the literature between old and modern wheat species. To help fill this gap, the baking quality characterization, gluten content, protein fraction composition, high molecular weight glutenin subunits, and rheological properties of ancient and modern wheat were determined and compared. These varieties were collected by the gene bank of the Crop Research Institute in Prague-Ruzyne and were grown in organically certified research areas in the Czech Republic. Results revealed differences in protein content and composition between varieties with different ploidy levels, as well as differences in development time and stability between einkorn and bread wheat varieties. Based on the proximity of their positions to the parameter quality in the principal components analysis, such as gluten content, gluten index (GI), Zeleny test, stability, dough development time (C1) and gliadin, the baking performances of cultivars were identified.