Xylella fastidiosa (Wells) is a xylem-limited bacterium that causes Pierce's disease of grapevines. The bacterium is transmitted by insect vectors such as the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). Experiments were conducted to compare the role of selected X. fastidiosa genes on 1) bacterial acquisition and retention in GWSS foreguts, and 2) transmission to grapevines by GWSS. Bacterial genotypes used were: mutants Xf-Delta pilG, Xf-Delta pilH, Xf-Delta gacA, and Xf-Delta popP; plus wild type (WT) as control. Results showed that Xf-Delta pilG had enhanced colonization rate and larger numbers in GWSS compared with WT. Yet, Xf-Delta pilG exhibited the same transmission efficiency as WT. The Xf-Delta pilH exhibited poor acquisition and retention. Although initial adhesion, multiplication, and retention of Xf-Delta pilH in GWSS were almost eliminated compared with WT, the mutation did not reduce transmission success in grapevines. Overall, Xf-Delta gacA showed colonization rates and numbers in foreguts similar to WT. The Xf-Delta gacA mutation did not affect initial adhesion, multiplication, and long-term retention compared with WT, and was not significantly diminished in transmission efficiency. In contrast, numbers of Xf-Delta popP were variable over time, displaying greatest fluctuation from highest to lowest levels. Thus, Xf-Delta popP had a strong, negative effect on initial adhesion, but adhered and slowly multiplied in the foregut. Again, transmission was not diminished compared to WT. Despite reductions in acquisition and retention by GWSS, transmission efficiency of genotypes to grapevines was not affected. Therefore, in order to stop the spread of X. fastidiosa by GWSS using gene-level targets, complete disruption of bacterial colonization mechanisms is required.