Background: Somatically acquired mitochondrial DNA (mtDNA) mutations have been linked to aging, degenerative diseases, cancer, and organ dysfunction. We investigated the mtDNA alterations in human atrial tissues from patients with chronic atrial fibrillation (AF). Materials and Methods: Left atrial appendages were obtained from patients undergoing open heart surgeries such as mitral/aortic valve replacement with written consent. Tissue samples were taken from 4 patients with chronic AF and from 2 matched patients with no history of AF and normal sinus rhythm. Total DNA was extracted from tissues and corresponding blood cells. In order to amplify and sequence the control region, cytochrome c oxidase (CO) I, COII, ATP synthase, and cytochrome b of mtDNA, we used 9 primer sets, the ABI Prism 3100 Genetic Analyzer with the BigDye Terminator v3.1 Ready Reaction Kit (Applied Biosystems). In an attempt to investigate mtDNA length heteroplasmy in the nucleotides (n)303 - 315, n16184 - 16193 poly-C regions, and CA repeats starting at n514, we carried out a qualitative and quantitative profiling of mtDNA length heteroplasmy using size-based PCR product separation by capillary electrophoresis (ABI 3100 and ABI Prism Genotyper version 3.1). Results: Seven mtDNA mutations were found, which had not previously been recorded in either published or unpublished mtDNA polymorphism databases (www.mitomap.org) and matched control group. We found two types of mtDNA alterations - base substitutions and small deletions/insertions as well as the length heteroplasmies of the poly-C regions in the control segments. Two patients (50%) had tissue-specific length heteroplasmic mutations from n16184 - 16193 poly-C tract and CA repeats starting at n514 respectively. Interestingly, a 9-bp deletion (nucleotide 8272-8281) around mtDNA COII gene was only found in tissues and corresponding blood cells from two patients. Conclusion: These findings strongly suggest that mtDNA mutations may play a crucial role in atrial dysfunction in patients with chronic AF.