Laboratory experiments were conducted to characterize the trichome-based defense of glandular-haired alfalfa, Medicago sativa L., against the potato leafhopper, Empoasca fabae (Harris). Within-plant variability in leafhopper resistance was examined by caging adult leafhoppers to either basal or apical stem internodes of the leafhopper-resistant, glandular-haired M. sativa genotype G98A or the susceptible, nonglandular-haired M. sativa ‘Ranger’. Young, actively secreting glandular trichomes are located on apical internodes of G98A, whereas senesced gland heads are found on older, basal internodes of G98A. After 96 h, the highest cumulative leafhopper mortality and lowest number of excretory droplets were associated with apical internodes of G98A. No difference was detected in mortality and feeding levels among insects caged to basal internodes of G98A and basal and apical internodes of Ranger. The influence of abiotic factors on leafhopper resistance was evaluated by caging adult leafhoppers to either G98A or Ranger under four combinations of low and high light (250 and 1,000 μmol s⁻¹ m⁻²) and temperature regimes (17 and 30°C). After 96 h, the highest cumulative mortality was associated with leafhoppers confined to G98A under high light and high temperature conditions. Temperature level and plant type also had an effect on the production of excretory droplets, resulting in the highest number of excretory droplets being associated with Ranger under the high temperature regime. These results indicate that certain regions of M. sativa G98A are better protected against the potato leafhopper than others and that temperature influences resistance levels of glandular-haired alfalfa.