Pierce’s disease is a major threat to the California grape industry. The disease-causing bacterium Xylella fastidiosa is vectored by a number of leafhoppers including Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Experiments were conducted to study H. vitripennis preference, feeding, and survivorship in response to four Vitis genotypes. Plants of V. vinifera (‘Chardonnay’), V. girdiana, V. candicans, and a V. rupestris × V. arizonica/candicans hybrid (D8909-17) were grown in pots in the greenhouse and transferred to laboratory conditions for experiments with field-collected H. vitripennis. A choice test without prior insect acclimation on grapes revealed that H. vitripennis selected Chardonnay over V. candicans throughout the duration of the experiment, whereas a shift in preference between D8909-17 and V. girdiana was observed over time. In a second set of choice tests, which were preceded by an acclimation on one of the four grape genotypes, significant genotype, time, and acclimation × genotype effects were observed. Chardonnay was preferred over V. candicans independent of acclimation genotype. Although H. vitripennis confined on D8909-17 excreted 1.8-fold (dry-weight corrected) the amount of insects feeding on V. candicans, differences in the rate of excreta production per insect or insect dry weight were not significant among grape genotypes. Adult mortality was greatest on V. candicans when H. vitripennis were confined in parafilm sachets for excreta collection as well as in a no-choice test. Grape genotype affected the behavior of adult H. vitripennis under controlled conditions, which may influence Pierce’s disease epidemiology under field conditions.