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Metallothioneins (MTs) belong to a group of small cysteine-rich proteins that are ubiquitous throughout all kingdoms. The main function of MTs is scavenging of free radicals and detoxification and homeostating of heavy metals. In humans, 16 genes localized on chromosome 16 have been identified to encode four MT isoforms labelled by numbers (MT-1–MT-4). MT-2, MT-3 and MT-4 proteins are encoded by a single gene. MT-1 comprises many (sub)isoforms. The known active MT-1 genes are MT-1A, -1B, -1E, -1F, -1G, -1H, -1M and -1X. The rest of the MT-1 genes (MT-1C, -1D, -1I, -1J and -1L) are pseudogenes. The expression and localization of individual MT (sub)isoforms and pseudogenes vary at intra-cellular level and in individual tissues. Changes in MT expression are associated with the process of carcinogenesis of various types of human malignancies, or with a more aggressive phenotype and therapeutic resistance. Hence, MT (sub)isoform profiling status could be utilized for diagnostics and therapy of tumour diseases. This review aims on a comprehensive summary of methods for analysis of MTs at (sub)isoforms levels, their expression in single tumour diseases and strategies how this knowledge can be utilized in anticancer therapy.