Mutations in the mitochondrial genome are associated with multiple diseases and biological processes; however, little is known about the extent of sequence variation in the mitochondrial transcriptome. By ultra-deeply sequencing mitochondrial RNA (>6000x) from the whole blood of similar to 1000 individuals from the CARTaGENE project, we identified remarkable levels of sequence variation within and across individuals, as well as sites that show consistent patterns of posttranscriptional modification. Using a genome-wide association study, we find that posttranscriptional modification of functionally important sites in mitochondrial transfer RNAs (tRNAs) is under strong genetic control, largely driven by a missense mutation in MRPP3 that explains similar to 22% of the variance. These results reveal a major nuclear genetic determinant of posttranscriptional modification in mitochondria and suggest that tRNA posttranscriptional modification may affect cellular energy production.