In the global action for biodiversity conservation, the most urgent need is to delimit and identify species. DNA barcoding and metabarcoding based on molecular markers have been increasingly used in species delimitation and species diversity assessment, respectively, owing to their advantages of standardization and high throughput. The molecular markers used in these methods for animals are mainly derived from mitochondrial DNA. The principle involved is that inter-specific genetic distances are greater than intra-specific genetic distances. However, if multiple mitochondrial haplotypes exist within individual specimens and the divergence between these haplotypes is greater than the assumed maximum intra-specific divergence, the species delimitation and species diversity assessment may be affected. In our recent studies, a widespread phenomenon of multiple mitochondrial haplotypes within individual specimens was discovered in fig wasps (Hymenoptera, Insecta), having negative impact on DNA barcoding and metabarcoding. In this study, we expanded the taxa to include up to 480 specimens from 82 species, 66 genera, 45 families, and 13 orders of Insecta and Arachnida. By high-throughput sequencing of mitochondrial cox1 fragments within individual specimens, we studied the pattern of multiple mitochondrial haplotypes within individual specimens. Three DNA metabarcoding strategies, one of which was simulated, were applied. The results revealed that multiple mitochondrial haplotypes within individual specimens could lead to an overestimation of species diversity by metabarcoding. Simultaneously, we found that the infection with intracellular endosymbiotic bacteria Wolbachia was significantly negatively related with this effect. These results suggest that additional attention should be paid to the interference of multiple mitochondrial haplotypes within individual specimens on the results of DNA metabarcoding in species diversity assessment of animals.