The Cambridge reference sequence (CRS) for human mitochondrial DNA (mtDNA) has been a cornerstone in studies of human evolution, population genetics, and mitochondrial diseases. However, discrepancies between the CRS and other mtDNA sequences have been noted, which may result from errors in the original sequencing or rare polymorphisms. The original sequence was primarily derived from a single individual of European descent, with some sequences from HeLa and bovine mtDNA. To address these issues, the authors resequenced the original placental mtDNA sample and identified both errors and rare polymorphisms in the CRS. They found 11 incorrect nucleotide positions and seven rare polymorphic alleles. The revised CRS mtDNA belongs to European haplogroup H, based on specific nucleotide substitutions. The overall error frequency for the original CRS analysis was 0.07%. The authors recommend correcting the ten simple substitution errors, retaining the rare polymorphic alleles, and maintaining the original nucleotide numbering to ensure accuracy and consistency.The Cambridge reference sequence (CRS) for human mitochondrial DNA (mtDNA) has been a cornerstone in studies of human evolution, population genetics, and mitochondrial diseases. However, discrepancies between the CRS and other mtDNA sequences have been noted, which may result from errors in the original sequencing or rare polymorphisms. The original sequence was primarily derived from a single individual of European descent, with some sequences from HeLa and bovine mtDNA. To address these issues, the authors resequenced the original placental mtDNA sample and identified both errors and rare polymorphisms in the CRS. They found 11 incorrect nucleotide positions and seven rare polymorphic alleles. The revised CRS mtDNA belongs to European haplogroup H, based on specific nucleotide substitutions. The overall error frequency for the original CRS analysis was 0.07%. The authors recommend correcting the ten simple substitution errors, retaining the rare polymorphic alleles, and maintaining the original nucleotide numbering to ensure accuracy and consistency.