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Background: Pearl millet (Pennisetum glaucum L.) has become increasingly attractive due to its health benefits. It is grown as food for human consumption and fodder for livestock in Africa and Asia. This study focused on five pearl millet populations from different agro-ecological zones from Tunisia, and on characterization by morphological traits, total phenolic and flavonoid content, antioxidant activity, and occurrence of Fusarium.Results: Analysis of variance revealed highly significant differences between populations for the quantitative traits. The highest grain weights occurred in the pearl millet cultivated in Zaafrana and Gergis of Tunisia. Early flowering and early maturing populations cultivated in the center (Zaafrana, Rejiche) and south (Gergis) of Tunisia tended to have a higher grain yield. The Zaafrana population showed the highest value of green fodder potentiel (number and weight of leaves/cultivar and the weight of tillers and total plant/cultivar) followed by Gergis and Rejiche. The Kelibia population showed the highest total phenolic and flavonoid content. Rejiche exhibited the greatest antioxidant activity. Trans-cinnamic, protocatechuic, and hydroxybenzoic acids were the major phenolic compounds 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 occurred most frequently. The average incidence of the three Fusarium species was relatively low (<5%) in all populations. The lowest infection rate (0.1%) was recorded in the samples from Zaafrana.Conclusion: Chemometric analysis confirmed the usefulness of the above traits for discrimination of pearl millet populations, where a considerable variation according to geographical origin and bioclimatic conditions was observed.