The Cambridge reference sequence (CRS) for human mitochondrial DNA (mtDNA) has been reanalyzed and revised. The original CRS, derived from a single European individual and containing some HeLa and bovine mtDNA sequences, was found to have errors and rare polymorphisms. Resequencing confirmed these discrepancies, including errors in nucleotide positions, such as a CC doublet actually being a single cytosine. The CRS also contains rare polymorphic alleles, some of which are private. The HeLa mtDNA sequence was found to contribute to one error, while bovine mtDNA contributed to errors at positions 14,272 and 14,365. The revised CRS, with corrected errors and retained rare polymorphisms, is assigned to haplogroup H. The original nucleotide numbering is retained to avoid confusion. The revised CRS should correct ten simple substitution errors, retain rare polymorphisms, and maintain the original numbering. The study highlights the importance of revising the CRS due to its widespread use. The resequencing was done using overlapping PCR fragments and alternative methods, yielding consistent results. The authors acknowledge the contributions of colleagues and funding sources. The revised CRS aims to be a true reference sequence, not a consensus sequence, ensuring accuracy and consistency with existing literature. The study emphasizes the need for careful revision of the CRS to improve its reliability for research in human evolution, population genetics, and mitochondrial diseases.The Cambridge reference sequence (CRS) for human mitochondrial DNA (mtDNA) has been reanalyzed and revised. The original CRS, derived from a single European individual and containing some HeLa and bovine mtDNA sequences, was found to have errors and rare polymorphisms. Resequencing confirmed these discrepancies, including errors in nucleotide positions, such as a CC doublet actually being a single cytosine. The CRS also contains rare polymorphic alleles, some of which are private. The HeLa mtDNA sequence was found to contribute to one error, while bovine mtDNA contributed to errors at positions 14,272 and 14,365. The revised CRS, with corrected errors and retained rare polymorphisms, is assigned to haplogroup H. The original nucleotide numbering is retained to avoid confusion. The revised CRS should correct ten simple substitution errors, retain rare polymorphisms, and maintain the original numbering. The study highlights the importance of revising the CRS due to its widespread use. The resequencing was done using overlapping PCR fragments and alternative methods, yielding consistent results. The authors acknowledge the contributions of colleagues and funding sources. The revised CRS aims to be a true reference sequence, not a consensus sequence, ensuring accuracy and consistency with existing literature. The study emphasizes the need for careful revision of the CRS to improve its reliability for research in human evolution, population genetics, and mitochondrial diseases.