Objective To explore the feasibility of detecting maternal hereditary mitochondrial tRNAThr15927G>A (m.15927G>A) mutation using buccal swabs. Methods We performed sequence analysis of mitochondrial DNA in blood samples from 2070 cases of maternal hereditary mitochondrial disease in the First Affiliated Hospital of Wenzhou Medical University, and identified 3 patients with m.15927G>A mutation. Buccal swabs and blood samples were obtained from the 3 patients (mutation group) and 3 normal volunteers (control group). After extracting whole genomic DNA from all the samples, the DNA concentration and purity were analyzed. The PCR products were subjected to dot blot hybridization, Southern blot hybridization, and DNA sequencing analysis to verify the feasibility of detecting m.15927G>A mutation using buccal swabs. Results There was no significant difference in DNA concentration extracted from buccal swabs and blood samples in either the mutation group or the control group (P>0.05), but the purity of manually extracted oral mucosa DNA was significantly lower than that of whole blood and oral mucosa DNA extracted using commercial kits (P<0.05). Dot blot hybridization and Southern blot hybridization both yielded positive results in the control group but negative results in the mutation group. DNA sequencing identified m.15927G>A mutation in all the samples from the mutation group. Conclusion Buccal swabs collection accurate is an accurate and sensitive method for the detection of m.15927G>A mutation.