A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNA^Tyr molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during the aminoacylation process. The impact of these substitutions on mt-tRNA^Tyr tyrosylation and structure was investigated using the corresponding tRNA transcripts. In vitro tyrosylation efficiency is decreased 600-fold for mutant A22G (mitochondrial gene mutation T5874C), 40-fold for G15A (C5877T), and is without significant effect on U54C (A5843G). Comparative solution probings with lead and nucleases on mutant and wild-type tRNA^Tyr molecules reveal a greater sensitivity to single-strand specific probes for mutants G15A and A22G. For both transcripts, the mutation triggers a structural destabilization in the D-loop that propagates toward the anticodon arm and thus hinders efficient tyrosylation. Further probing analysis combined with phylogenetic data support the participation of G15 and A22 in the tertiary network of human mt-tRNA^Tyr via nonclassical Watson–Crick G15–C48 and G13–A22 pairings. In contrast, the pathogenic effect of the tyrosylable mutant U54C, where structure is only marginally affected, has to be sought at another level of the tRNA^Tyr life cycle.