Objective To explore the mutation situation of pancreatic cancer cell mitochondria DNA D-loop region. Method PCR and direct sequencing was used to analyze the mutational site of mitochondria DNA D-loop region in two pancreatic cancer cell lines SW1990 and JF-305 and normal primary cultured pancreas cell. Result The point mutations were found in two pancreatic cancer cell lines and normal primary cultured pancreas cell. Eight point mutations were found in SW1990 and 9 point mutations were found in JF-305. Three point mutations (73 site A-G,16223 site C-T, and 16358 site c-T) existed in all two pancreatic cancer cell lines and normal primary cultured pancreas cells, which can be considered as polymorphism. Other two point mutations (16211 site C-T and 16311 site T-C) were only found in two pancreatic cancer cell lines, which can be considered as special mutations. Conclusion The mitochondrial DNA D-loop region of pancreatic cancer cells existed polymorphism and special mutations, and the special mutations might be new molecule marker.