The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a cosmopolitan polyphagous aphid species that inflicts serious damage to a wide spectrum of crops and has an extraordinary ability to transmit viruses in either a nonpersistent or persistent mode. It tends to specialize on a few host plants species, resulting in parapatric divergence among populations in a given geographical area over a period of time. This seems to be the major reason for its remarkable diversity resulting in the evolution of biotypes and cryptic species favoring host adaptation and reproductive isolation across various localities. This notorious pest is supposed to have a number of putative biotypes associated with hosts or geographical locations. It is suspected that A. gossypii occurring on cotton (Malvaceae) and melons (Cucurbitaceae) are different biotypes. The relationship between host or geographic preference and genetic variation of aphids is unclear and thus requires further investigation. In light of this, the present study was conducted to determine if host- or geographical location-associated genetic differences are indeed present in 116 populations of A. gossypii collected from various agroclimatic zones of India on various host plants with special emphasis on Malvaceae and Cucurbitaceae families using the well-known mitochondrial cytochrome oxidase I (mtCOI) marker. Alignment of mtCOI sequences of A. gossypii along with available sequences in the National Center for Biotechnology Information (NCBI), GenBank, and Barcode of Life Data (BOLD) system revealed maximum sequence identity of 99% with very few variable sites indicating an extremely low level of intraspecific variation. The phylogram strongly suggested that there is no major host- or geographical location-associated genetic differences in A. gossypii, and it is a single cosmopolitan species devoid of genetic variations. This study aids to resolve evolutionary relationships among closely related species groups and detect newly evolving biotypes.