Introduction of the glassy-winged sharpshooter (GWSS) into California has caused an increase in incidence of Pierce’s Disease in grape-growing regions. Although host plant resistance to the causative bacterium, Xylella fastidiosa, and/or its GWSS vector is being sought, research is hampered by lack of understanding of the transmission process. X. fastidiosa (Xf) is acquired into and colonizes the anterior foregut of the vector (the precibarium and cibarium), and is somehow inoculated back into the plant from this area during the feeding process. To study the location of acquisition (from which inoculation occurs), we developed a confocal laser scanning microscopy method to visualize green fluorescent protein (GFP)-transformed Xf in situ within the undissected foregut of the vector. Fine structure of the colonies was then examined via scanning electron microscopy. We describe here the first time course study of GFP-Xf acquisition by GWSS, across varying acquisition access periods. Bacterial colonies first form in the precibarium, then later accumulate in the cibarium. Implications for inoculation of bacteria from these sites are discussed.