Species belonging to the genus Populus (poplars) produce a series of defensive proteins in response to insect damage. Proteinase inhibitors, polyphenol oxidases, and chitinases are the most relevant and intensively studied proteins. Most of the knowledge about the relation between these proteins and herbivores has been obtained from studies with chewing insects. Nothing is known about whether phloem-feeder insects such as aphids are able to trigger a comparable response. In the current study, the expression of genes encoding a Kunitz trypsin inhibitor 3 (KTI3), a polyphenol oxidase 1 (PPO1), and a class I chitinase (CHI) was characterized in two poplar hybrids (one resistant hybrid and one susceptible hybrid, to aphids) attacked by the aphid Chaitophorus leucomelas Koch. The expression pattern was analyzed using a semiquantitative reverse transcription-polymerase chain reaction approach. The expression of KTI3 was increased by aphids only in the aphid-susceptible hybrid. Differently, PPO1 expression was increased by aphids in the aphid-resistant hybrid. The expression of CHI was down-regulated by aphids in the susceptible hybrid. This is the first study to report the differential expression of poplar defense genes in response to phloem-feeder insects such as aphids. The findings from the current study suggest that the expression levels of defensive proteins are affected by poplar genotype and by aphid infestation.