OBJECTIVE To establish a rapid, accurate, noninvasive and low cost method for screening MT3243A>G mutation in mitochondrial diabetes. METHODS Blood, saliva, and urine sediment samples were collected from 6 patients with confirmed mitochondrial diabetes and 50 healthy controls from Shanghai Children's Hospital and Shanghai Sixth People's Hospital. The heterozygosity levels of MT3243A>G mutation in above samples were detected with pyrosequencing, and the data were compared. MT3243A>G mutations were rapidly screened with high resolution melting curve analysis (HRM) in the urine sediment samples of 1070 diabetic patients from 4 communities in Shanghai. Furthermore, pyrosequencing was used to validate the suspected positive samples, and the heterozygosity levels were also quantified. RESULTS Comparative experiments found that heterozygosity of MT3243A>G mutation was 2 to 7 times higher in urine sediment than in saliva and blood samples from the 6 patients with confirmed mitochondrial diabetes. However, the heterozygosity was slightly higher in saliva than blood samples. MT3243A>G mutation was not detected in the 50 healthy controls. Two samples with suspected MT3243A>G mutation were identified in the 1070 urine sediment samples of diabetes patients with HRM screening, which were validated by pyrosequencing. The heterozygosity of MT3243A>G mutation were 33.32% and 14.67% in the urine sediment samples, respectively. CONCLUSION Urine sediment samples can be used for rapid screening of MT3243A>G mutation for its ease to collect, noninvasiveness and higher level of heterozygosity. HRM is suitable for rapid screening for mitochondrian mutations for its low cost, while such mutations could be detected with sensitivity and accuracy by pyrosequencing.