Mitochondrial single nucleotide polymorphisms (mtSNPs) have been reported to associate with type-2 diabetes mellitus (T2DM), but mtSNPs appear to be considerably different among different populations and regions. To determine mtSNPs in Chinese Han patients with T2DM, the entire sequences of the mitochondrial genomes from 72 T2DM Chinese (59 +/- 4 years) and 50 age-matched healthy subjects (controls) in Chongqing region of Western China were directly sequenced and mtSNPs were analyzed. We found that M8, M9, D, G, R and A haplogroups exist in Chinese Han population and the frequency of haplogroup M9 was significantly higher in patients with T2DM than in the controls (p = 0.0006, OR 0.06 [95% CI 0.008-0.476]). MtSNPs T3394C in NADH dehydrogenase subunit 1 (ND1), G4491A in ND2, T16189C and T16519C were found with significantly higher frequency in patients with T2DM than in the controls (T16189C, p = 0.0045; T16519C, p < 0.0001; T3394C, p = 0.0015; G4491A, p = 0.0015). In contrast, the frequency of C5178A in ND2 and A10398G in ND3 was higher in the controls than in patients with T2DM (C5178A, p = 0.014; A10398G, p = 0.0011). Our results indicate that mtSNPs T3394C, G4491A, T16189C and T16519C show susceptible tendency to T2DM and mtSNPs C5178A and A10398G seem to be genetic factors for against T2DM. These mtSNPs determined in our study is useful and could be used for early diagnosis and prevention of T2DM in Chinese Han population.