Pearl millet (Pennisetum glaucum L.) has become increasingly attractive due to its health benefits. It's grown for food and fodder in Africa and Asia for human consumption and fodder for livestock. In this study, our work focused on five pearl millet populations from different agro-ecological zones from Tunisia on characterization in morphological traits, total phenolic and flavonoid contents, antioxidant activity and Fusarium occurrence.Variance analysis revealed highly significant differences between populations for the quantitative traits. The highest weights' values were achieved from the pearl millet cultivated in Zaafrana and Gergis of Tunisia. Moreover, early-flowering and early maturing populations cultivated in the center (Zaafrana, Rejiche) and south (Gergis) of Tunisia tended to have a higher grain yield. Zaafrana population showed the highest value of green fodder potential followed by Gergis and Rejiche. Kelibia population showed the presence of the maximum amount of total phenolic and flavonoid contents. Rejiche exhibited maximum antioxidant activity. Trans-cinnamic, protocatechuic and hydroxybenzoic acids were the major phenolic compound in all the extracts. Three Fusarium species were identified in Tunisian pearl millet populations based on morphologic and molecular characterization. Fusarium graminearum and Fusarium culmorum had the most predominant frequencies. The average incidence of the three Fusarium species was relatively low (< 5%) in all populations, where the least infection rate (0.1%) was recorded in Zaafrana samples.Chemometric analysis confirmed the usefulness of the above traits for discrimination of pearl millet populations, where a considerable structure according to geographical origin and bioclimatic conditions was observed. This article is protected by copyright. All rights reserved.