Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and beta-amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and beta-amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, beta-amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.