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The Ocimum genus is one of Indiaʼs prominent botanical classes of traditional medicinal culture comprising medicinally and agronomically important plants. Morphological resemblances, overlapping geographical distribution, and history of traditional nomenclature have necessitated a comprehensive qualitative report for effective quality control and removing the species ambiguity pertaining to this genus. This paper provides detailed morpho-micrometric characteristics used to differentiate between six indigenous Ocimum species of India. Among them, O. gratissimum was distinguished as the only shrub with a fleshy petiole. In green and purple forms, O. tenuiflorum leaves had serrate margins and showed no particular anatomical differences except for the anthocyanins containing epidermal cells of the latter. O. basilicum had glabrous leaves except for the veins, which were puberulous. O. filamentosum had tenuous anther filaments and was the least aromatic while O. africanum had a citrusy odour, which along with the number of xylary rows, size of mesophyll cells, and epidermal cell wall architecture, distinguished it from O. americanum. An HPTLC method was developed using experimental design and validated for quantification of multi-class compounds from terpenoic, phenolic acids, and flavonoids in Ocimum leaves. It was found linear (r 2 > 0.99) with recoveries between 95 – 100% for all compounds. The eluted bands of marker compounds were subjected to HPTLC-MS analysis as a confirmative tool. This is the first anatomical and analytical report of O. filamentosum Forssk. The obtained results could be effectively used for species identification using vegetative characters alone with the anatomical-HPTLC data backing up the former as a rapid and economical tool. [ABSTRACT FROM AUTHOR]