Aphis gossypii Glover and Acyrthosiphon gossypii Mordvilko (Hemiptera: Aphididae) are key pests of cotton, Gossypium hirsutum L., known to induce cotton host plant defense responses. Deep RNA sequencing of the cotton transcriptome followed by differential expression analyses were performed to clarify the molecular mechanisms of cotton defense in response to feeding by these aphid pests. We found 6,565 genes were differentially expressed in cotton in response to feeding by Ac. gossypii and 823 genes that were differentially expressed in response to feeding by A. gossypii, while 2,379 genes were differentially expressed in response to simultaneous feeding by both species. Pathway enrichment analysis showed that the differentially expressed genes associated with Ac. gossypii feeding were enriched for metabolic pathways, porphyrin and chlorophyll metabolism, biosynthesis of secondary metabolites, biosynthesis of carotenoids, and the pentose phosphate pathway. The enriched pathways in cotton fed on by A. gossypii were thiamine metabolism, glutathione metabolism, plant–pathogen interaction, and sesquiterpene and triterpenoid biosynthesis. The differentially expressed genes in cotton induced by simultaneous feeding of both species were primarily related to circadian rhythm regulation, photosynthesis, porphyrin and chlorophyll metabolism, galactose metabolism, and flavonoid biosynthesis.