Specific mitochondrial DNA (mtDNA) mutations in 12SrRNA and tRNASer(UCN) cause non-syndromic hearing loss (NSHL). In this study, we screened 466 hearing loss (HL) patients, negative for GJB2 mutations, for mutations in the two mtDNA genes and flanking regions. In total, 43 different variants were identified, 31 of which were polymorphisms, one was a mutation (m.1555A-->G), two were known variants of controversial pathological nature (m.827A-->G and m.961delTinsC(n)) and nine were newly identified variants. The frequency of m.1555A-->G in this set of HL patients was 0.3%, which was lower than expected. To assess the putative causative nature of controversial or newly identified variants, the frequencies of these variants were determined in 400 Belgian control subjects, and their effect on the secondary structure and their conservation among different species was determined. Our data provide further support for a polymorphic nature of the controversial m.961delTinsC(n) variant. In addition, two of the newly identified variants, m.636A-->G in the 12SrRNA flanking tRNA(Phe) and m.990T-->C in 12SrRNA, may be new candidates for pathogenic HL variants. If the pathogenic nature of m.636A-->G can be confirmed, this would be the first NSHL mutation in tRNA(Phe).