In the present study, the petiole anatomy of 39 species from tribes Cichorieae (18), Cardueae (10), Anthemideae (5), Inuleae (4), Gymnarrheneae (1), Astereae (1), and Gnaphalieae (1) was investigated to determine their taxonomic significance. Leica Light Microscope (Model 1000) embedded with the Infinity 1-5 C-MEL (Canada) digital camera was used to take the photomicrographs. Results revealed intra and interspecific variation. Among the examined traits, the significant systematic qualitative characteristics are petiole outline, arrangement and number of vascular bundles, shape and number of layers of different cells, and air spaces. The delimitation of taxa up to species level is carried via taxonomic key based on these qualitative traits. The quantitative data of length and width of the petiole, epidermal cells, chlorenchyma cells, parenchyma cells, vascular bundles, xylem vessels, and phloem cells, were statistically analyzed, collected into a matrix, and examined their significance in the species separation. Variations were observed in the petiole and vascular bundle size on the multivariate ordination of principal components. The highest positive correlation (0.87 - 0.98) is found in xylem vessels length and width with phloem cells length and width. The least positive correlation (0.07) was between vascular bundle length with xylem vessels and phloem cells. The two-way dendrogram on neighbor-joining clustering in multivariate analysis summarized that the selected quantitative traits are significant in distinguishing the examined species. Conclusively petiole anatomical traits are significant diagnostic tools that assist the separation of Asteraceous taxa.