To develop criteria to interpret mitochondrial transfer RNA (mt-tRNA) variants based on unique characteristics of mitochondrial genetics and conserved structural/functional properties of tRNA. We developed rules on a set of established pathogenic/benign variants by examining heteroplasmy correlations with phenotype, tissue distribution, family members, and among unrelated families from published literature. We validated these deduced rules using our new cases and applied them to classify novel variants. Evaluation of previously reported pathogenic variants found that 80.6% had sufficient evidence to support phenotypic correlation with heteroplasmy levels among and within families. The remaining variants were downgraded due to the lack of similar evidence. Application of the verified criteria resulted in rescoring 80.8% of reported variants of uncertain significance (VUS) to benign and likely benign. Among 97 novel variants, none met pathogenic criteria. A large proportion of novel variants (84.5%) remained as VUS, while only 10.3% were likely pathogenic. Detection of these novel variants in additional individuals would facilitate their classification. Proper interpretation of mt-tRNA variants is crucial for accurate clinical diagnosis and genetic counseling. Correlations with tissue distribution, heteroplasmy levels, predicted perturbations to tRNA structure, and phenotypes provide important evidence for determining the clinical significance of mt-tRNA variants.