Pathotype differentiation within Xylella fastidiosa (Xf) is necessary to accurately identify causal agents of leaf scorch diseases in agronomic crops and ornamentals. Three strain groups, Pierce’s disease (PD), almond leaf scorch (ALS), and oleander leaf scorch (OLS), occur in California and inhabit multiple hosts with varied severity. A SYBR® Green-based quantitative real time PCR (QRT PCR) protocol with a single primer set was developed for Xf genotype differentiation utilizing melting temperature (Tm) analysis. This system separated PD, ALS, and OLS strains from one another similar to the placement in previous reports. Influence of environmental background on Tm of PCR product was reduced by standardizing DNA extraction protocols and the master mix for PCR. With this system, Xf was detected and identified to strain in potential insect vectors.