Molecular markers are useful tools for measuring the genetic diversity among agricultural species. In plants, microsatellites are still the most used markers for germplasm characterization, conservation, and traceability purposes, while in the livestock sector, although having represented the standard for at least two decades, they are still used only for minor farm animal species. In this work, together with a review on the use of microsatellites in livestock, we also illustrate the use of these markers for the characterization of agricultural diversity and food traceability through two case studies: (i) the analysis of genetic diversity in ancient fruit tree cultivars of apple (Malus × domestica Borkh.), pear (Pyrus communis L.), sweet cherry (Prunus avium L.), and sour cherry (Prunus cerasus L.) from Northern Italy and (ii) the molecular authentication of wheat food chain. In the former case, a high genetic variability as well as the presence of different ploidy levels were detected, while in the latter microsatellite markers were shown to be useful for traceability and product authentication along the whole food chain. Overall, the presented evidence confirms the versatility of microsatellites as markers for both agrobiodiversity characterization and food traceability in cultivated plants and farm animals.