The presence of Xylella fastidiosa in the region of Apulia, Italy, represents one of the most serious plant-health emergencies of recent years that Italy and the entire European Union have had to deal with. The Italian Ministry of Agriculture transposed the European Union guidelines that define the necessary measures to prevent the introduction and spread of X. fastidiosa, and has organized a monitoring plan for preventive purposes across the entire national territory. In this study, we report the experience of the Latium region, Italy, as an example of a 'pest-free' area; 315 inspections were carried out for a total area of 416 ha. With the analysis mainly focused on asymptomatic plant material, the diagnosis was based on a molecular method characterized by high specificity and sensitivity: real-time PCR. This method allowed the presence of X. fastidiosa to be proved or excluded in the processed samples, even though visual examination occasionally revealed the presence of host plants with suspicious symptoms. An in-depth description of the adopted approaches is reported here, along with the advantages and disadvantages of this diagnostic method. New techniques, such as loop-mediated isothermal amplification of DNA (LAMP), can improve the procedures by maintaining high sensitivity and specificity, in view of the application of the analysis directly in the field.