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The biodiversity of genus Carduus in Bulgaria is grossly under-studied with modern methods, which leads to unclear status of species and hampers the assessment of their habitats and conservation status. In this study, we used the variability of internal transcribed spacers (ITS) to investigate the biodiversity of Carduus species collected from different floristic regions of Bulgaria. Thirty-three samples were processed. Among the studied species, C. acanthoides exhibited the highest ITS variability (eight single nucleotide polymorphisms were found). High ITS variability was also found in C. crispus and C. hamulosus, demonstrating that some of the local Carduus species possesses unique genetic diversity, which can serve as source for future divergence of new forms and subspecies. For each species, unique nucleotides suitable as molecular taxonomy markers were identified. However, C. nutans and C. thoermeri displayed completely identical ITS sequences, which is in agreement with all ITS sequences of these species collected elsewhere in the world and deposited in the NCBI database. After careful analyses of both molecular and morphological data, we propose to restore C. nutans and C. thoermeri as one species with two subspecies. Namely, Carduus nutans subsp. nutans and Carduus nutans subsp. thoermeri. We recommend diameter of capitulum, length of peduncle, width of peduncle and width of bract as diagnostic features of taxonomic significance for distinguishing the two subspecies. [ABSTRACT FROM AUTHOR]