Maternally inherited diabetes and deafness and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes result from the 3243A>G mitochondrial point mutation. Current methods to detect the presence of the mutation have limited sensitivity and may lead to potential misclassification of patients with low levels of heteroplasmy. Here, we describe development and validation of a rapid real-time polymerase chain reaction (PCR) method for detection and quantification of levels of heteroplasmy in a single assay. Standard curve analysis indicated that the sensitivity of detection was less than 0.1%. Time from sample loading to data analysis was 110 minutes. We tested 293 samples including 23 known positives, 40 known negatives, and 230 samples from patients clinically classified as having type 2 diabetes. All positive samples were correctly detected, and of those samples previously quantified, heteroplasmy levels determined using the real-time assay correlated well (r(2) = 0.88 and 0.93) with results from fluorescently labeled PCR-restriction fragment length polymorphism and pyrosequencing methods. Screening of 230 patients classified as having type 2 diabetes revealed one patient with 0.6% heteroplasmy who had previously tested negative by PCR-restriction fragment length polymorphism. Real-time PCR provides rapid simultaneous detection and quantification of the 3243A>G mutation to a detection limit of less than 0.1%, without post-PCR manipulation.