Pathotype differentiation within Xylella fastidiosa (Wells) is necessary to accurately identify the causal agents of several leaf scorch diseases in agronomic crops and ornamentals. Three strain groups, Pierce's disease (PD), almond leaf scorch (ALS), and oleander leaf scorch (OLS), have been described in California and inhabit multiple hosts with varied severity. An SYBR (R) Green-based quantitative real-time PCR (QRT-PCR) protocol with a single primer set was developed for X. fastidiosa genotype differentiation utilizing melting temperature (T-m) analysis. The goal of this work was to develop a rapid diagnostic tool that could separate X. fastidiosa strains from one another similar to the placement in previous reports. Placement of eight PD, six ALS, and six OLS strains into respective strain groups was consistent with T-m curve analysis using a single primer set. These groupings were consistent with published data on the phylogenetic placement of the strains.