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Medicinal plant tissue cultures are potential sources of bioactive compounds. In this study, we report the chemical characterization of the callus cultures of three medicinal <i>Tilia</i> spp. (<i>Tilia cordata</i>, <i>Tilia vulgaris</i> and <i>Tilia tomentosa</i>), along with the comparison to bracts and flowers of the same species. Our aim was to show that calli of <i>Tilia</i> spp. are good alternatives to the calli of <i>T. americana</i> for the production of polyphenols and are better sources of a subset of polyphenolic metabolites, compared to the original organs. Calli were initiated from young bracts and grown on woody plant medium containing 1 mg L⁻¹ 2,4-D and 0.1 mg L⁻¹ BAP. For chemical characterization, a quality-controlled untargeted metabolomics approach and the quantification of several bioactive compounds was performed with the use of LC-ESI-MS/MS. While bracts and flowers contained flavonoid glycosides (astragalin, isoquercitrin) as major polyphenols, calli of all species contained catechins, coumarins (fraxin, esculin and scopoletin) and flavane aglyca. <i>T. tomentosa</i> calli contained 5397 µg g DW⁻¹ catechin, 201 µg g DW⁻¹ esculin, 218 µg g DW⁻¹ taxifolin and 273 µg g DW⁻¹ eriodictyol, while calli from other species contained lower amounts. <i>T. cordata</i> and <i>T. tomentosa</i> flowers were rich in isoquercitrin, containing 8134 and 6385 µg g DW⁻¹, respectively. The currently tested species contained many of the bioactive metabolites described from <i>T. americana</i>. The production of catechin was shown to be comparable to the most efficient tissue cultures reported. Flowers and bracts contained flavonoid glycosides, including tiliroside, resembling bioactive fractions of <i>T. americana</i>. In addition, untargeted metabolomics has shown fingerprint-like differences among species, highlighting possible chemotaxonomic and quality control applications, especially for bracts.