Leber hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases, which is mainly caused by three mitochondrial DNA (mtDNA) mutations (m.3460G>A, m.11778G>A and m.14484T>C). Incomplete penetrance suggests that there might be asymptomatic carriers in general populations. These asymptomatic carriers are clinically important as they are potential future patients and the female carriers could transfer the pathogenic mutations to their offspring. Thus, screening the three LHON primary mutations in general populations is important for genetic counseling.We optimized a multiplex allele-specific PCR method based on previous studies, and the sensitivity was evaluated. The three LHON primary mutations were screened by using this MAS-PCR method in 1571 subjects from general Chinese populations that are without symptoms or family history of optic neuropathy.The optimized MAS-PCR approach can detect a heteroplasmy level at 5%, 5%, and 20% for m.3460G>A, m.11778G>A and m.14484T>C, respectively. None of the three LHON primary mutations was detected in the 1571 subjects.The three LHON primary mutations are rare in general Chinese populations. The optimized MAS-PCR assay provides an easier, faster and more cost-effective method for detection of the three LHON primary mutations, making it practical for clinical diagnosis.