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Giloy (Tinospora cordifolia (Willd.) Miers.) has been a potential medicinal plant since ancient times; even today, it has great economical values; however, it still receives less attention in cytogenetic study. Detailed baseline data of chromosomes in mitotic and meiotic cell division remain very important for genetical characterization and evaluation of the reproductive potentiality of a species. Cytogenetic characterization with the aid of a chromosome banding technique is beneficial in searching chromosomal landmarks and constructing an accurate karyotype, which is completely lacking in the genus Tinospora. Hence, this is the first attempt of the detailed karyological study with fluorescence banding after enzymatic degradation of cell wall. Chromosomes of T. cordifolia are small (1.8–2.4 µm) and 2n=26 having a symmetric karyotype. The secondary constriction of two submetacentric pairs has DAPI-/CMA+ bands. The meiosis studies of male flowers show the presence of 13 bivalents having secondary associations among themselves. Meiotic abnormalities such as precocious movement (7.56%) and laggard (2.83%) were recorded, and the pollen viability was estimated to be 47.17%. In addition to the genetical characterization, assessment of the therapeutic value is imperative for the medicinal plants. Hence, the berberine content produced in the stem of T. cordifolia has been quantitatively measured by high-performance liquid chromatography and found to be 0.424±0.02% on dry weight basis. In this study, precise karyological profiling by differential banding has been constructed and linked with the medicinal quality of the genotype thus, considered to be beneficial in the selection, cultivation, management, and improvement program of this species.