Populations of the aphid Chaitophorous populicola feed most frequently on the stems and lamina of new and rapidly expanding leaves, and on the petioles of senescing leaves of their host plant, cottonwood (Populus deltoides). This study’s aim was to determine which trophic factors best explain intra-plant feeding location of C. populicola on P. deltoides ST109 clones. Bottom-up factors associated with leaf developmental stage correlate with preferred and rejected aphid feeding locations. HPLC analysis of ST109 phloem sap revealed that new leaves have a higher content of phenolic glycosides and certain amino acids than other developmental stages. Mature leaves have higher concentrations of gamma-amino butyric acid and are least preferred by C. populicola. Increasing leaf lignification and distance from phytodermal surface to vascular bundle are inversely correlated with preferred aphid feeding sites. Conversely, top-down factors explain other aspects of aphid feeding distribution. Ladybug beetles do not appear to influence the pattern of aphid feeding location. However, the presence of ant mutualists correlates with occurrence of aphids on mature leaves, and ants appear to be attracted to feeding aphids on mature leaves because they produce more honeydew than on other leaf stage. Thus, bottom-up factors associated with leaf developmental stage, like leaf toughness and the phloem sap constituents, appear to play a dominant role, while top-down factors such as ant mutualist behavior play a subsidiary role in determining aphid feeding location.