This study aims to evaluate the bio-morphological and biochemical variability of three Tunisian wild populations and one growing cultivar of purslane (Portulaca oleracea L.). The studied varieties should be easily distinguished by the color and the habitus of the plant as mentioned in literature, but the various analyses have shown a strong morphological heterogeneity within and among the wild and cultivated accessions as presented by the variance analysis test (ANOVA) and the PCA (Principal component analysis). We found high intrapopulation variability through the wild populations that make it hard to differentiate them only on the base of morphology. We analyzed the biochemical profile of those populations based on the analysis of freeze-dried samples of leaves and stems. We identified and quantified twelve different phenolic compounds by the HPLC-diode array detector (DAD) technique. Six phenolic acids and flavonoids were identified in the leaves and stems of the wild and cultivated populations. Sinapic acid and myricetin are the majors identified compounds through our samples. The results were significantly different in relation to the plant organs and to the geographic origin for most of the compounds. The obtained results highlighted the importance of Portulaca as a medicinal plant by showing its richness in phenols and flavonoids that have multi-medicinal effects besides their antioxidant power.