Aminoglycosides are potent antibiotics which are used to treat severe gram-negative infections, neonatal sepsis, and multidrug-resistant tuberculosis. Ototoxicity is a well-known side effect of aminoglycosides, and a rapid, profound, and irreversible hearing loss can occur in predisposed individuals. MT-RNR1 gene encoding the mitochondrial ribosomal 12S subunit is a hot spot for aminoglycoside-induced hearing loss mutations, however, a variability in the nature and frequency of genetic changes in different populations exists. The objective of this study was to analyze MT-RNR1 gene mutations in a Baltic-speaking Latvian population, and to estimate the prevalence of such genetic changes in the population-specific mitochondrial haplogroups. In the cohort of 191 ethnic non-related Latvians, the presence of two deafness-associated mutations, m.1555A>G and m.827A>G, three potentially pathogenic variations, m.961insC(n), m.961T>G and m.951G>A, and one unknown substitution, m961T>A was detected, and the aggregate frequency of all variants was 7.3%. All genetic changes were detected in samples belonged to the haplogroups H, U, T, and J. The presence of several aminoglycoside ototoxicity-related MT-RNR1 gene mutations in Baltic-speaking Latvian population indicates the necessity to include ototoxicity-related mutation analysis in the future studies in order to determine the feasibility of DNA screening for patients before administration of aminoglycoside therapy.