Hoverflies are potential candidates for biological control of aphid populations; however, identification of closely related hoverfly species by using morphological characteristics is quite difficult. For instance, adults of Sphaerophoria macrogaster (Thomson) (Diptera: Syrphidae)—the predominant species early in the vegetable production season in Japan—are morphologically indistinguishable from those of Sphaerophoria indiana Bigot (Diptera: Syrphidae) without comparison of the male genitalia. Here, we investigated genetic variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene of the two species by DNA sequencing and developed a multiplex PCR method for differentiating the two species. Alignment of COI sequences revealed 1.2% nucleotide variance between the two species. The COI sequence of S. macrogaster collected from Japan was 99.8% identical to those of S. macrogaster collected from India. The COI sequence of S. indiana collected from Japan was 100% identical to that of Sphaerophoria philanthus (Meigen) (Diptera: Syrphidae) collected from Canada. The sizes of the multiplex PCR products differentiated following gel electrophoresis were 162 bp for S. macrogaster and 607 bp for S. macrogaster and S. indiana. The accuracy rate of multiple PCR was 100%. Use of this method will facilitate further research into the characteristics of hoverflies and will improve the efficacy of biological control using hoverflies on vegetable crops.