In these studies we tested the effects of feeding substrate on the acquisition and retention of the bacterial plant pathogen Xylella fastidiosa by the glassy-winged sharpshooter (GWSS), Homalodisca coagulata. Grape plants (Vitis spp.) infected with a Pierce’s disease (PD) strain of X. fastidiosa, and oleander plants (Nerium oleander) infected with an oleander leaf scorch (OLS) strain were used as sources of inoculum. The strain of X. fastidiosa infecting plants was confirmed by PCR. Groups of GWSS adults were caged on either an OLS infected oleander plant, or a PD infected grapevine for 2 days. Insects from each source plant species were split into two groups, and moved to an uninfected plant of the same species as the source plant (oleander or grape), or to a non-host of either bacterial strain (chrysanthemum). Samples of insects were collected at 1, 3, and 7 days after transfer to uninfected hosts and tested using strain-specific PCR methods to detect the pathogen. We found that the proportion of insects retaining a host-specific strain was similar, regardless of whether insects were subsequently fed on a host or non-host of that bacterial strain. Future studies will test the effects of varying insect feeding substrate on the transmission rates of each strain.