Synonymous codon changes may not always be neutral indicating their significance in disease association studies, which is almost always overlooked. Synonymous substitutions may affect protein-folding rates leading to protein misfolding and aggregation. Genome wide analysis of 2301 mitochondrial genomes is performed to evaluate the significance of synonymous codons in disease association studies. The analysis revealed usage of rare codons at several sites in mitochondrial genes with rare codon usage higher for hydrophobic amino acids. The analysis suggests that variation data in association studies should be analyzed using site-specific codon usage values to infer the potential phenotypic impact of synonymous changes.