Few studies have explored the differences between Sanger and HTS methods in the results of mitogenome sequencing. We used a single individual of insect to study the differences between the sequences given by Sanger and PCR-free HTS methods. Here we provided evidence for biased results of sequencing due to different methods in the mitochondrial genes of atp6, atp8, cox1, cox2, cox3, Cytb, nad2, nad3, nad4, nad5, rrnS, rrnL, trnH, trnI, and control region at various degrees. Especially, in cox1, the differently sequenced nucleotides account for 2.6% of the complete length. Furthermore, the highest value of the intraspecific genetic distance based on K2P accounts for 2.5% using a barcode fragment size of cox1 (651 bp, Sanger), while the maximum distance of the corresponding cox1 fragment obtained by the two sequencing methods was 5.0%. We revealed that the methods of Sanger and HTS may give different sequencing results of mitochondrial genes, which may reflect the heteroplasmy of mitogenomes within an insect individual. Therefore, researchers should be very cautious in using the mixed data of a gene given by different methods of sequencing.