Aphids (Hemiptera: Aphidoidea: Aphididae) have distinctive features and significant variability in interesting biological traits. Aphididae comprises c. 5000 species distributed worldwide with a rapid diversification rate, which makes reliable phylogenetic reconstruction difficult as a framework for evolutionary inference. Despite several studies, the phylogenies of various subfamilies of Aphididae are not yet resolved, and their phylogenetic positions are still debated. Our study, based on two mitochondrial markers (COI and COII) and one nuclear marker (EF-1α), provided confirmation of some of the discrepancies as well as concordances of previous work, and thus contributes to an understanding of the higher-level relationships of Aphididae. Analyses of the combined dataset produced a well-resolved phylogenetic tree in which the major ten subfamilies of aphids correspond well with their associations with host plants. Sister relationships were found between Aphidini and Macrosiphini (Hormaphidini + Nipponaphidini) and Cerataphidini, and Fordini and Pemphigini. Lachnini is the only non-monophyletic tribe and Lizeriinae, represented by Paoliella nirmalae, occupied the basal position in Aphididae. Molecular dating of divergence revealed that diversification among the tribes and subtribes occurred in the Late Cretaceous to Late Oligocene. Thus, our phylogenetic analysis provides further insights into understanding the higher-level relationships within Aphidoidea.