Leifsonia xyli subsp xyli (Lxx) is the etiological agent of ratoon-stunting disease (RSD), one of the most important diseases that affects productivity of sugarcane crops. The lack of defined external symptoms in most plants is a challenge for the diagnosis of RSD facilitating its spread in production fields. In this study a Taqman® real-time polymerase chain reaction (qPCR) assay was developed for detection of the bacteria in different tissues of the plants and compared to conventional PCR and nested-PCR assays. The bacterial DNA was amplified by the nested-PCR and qPCR assays, indicating that conventional PCR lacks sufficient sensitivity to be used as a diagnostic tool for RSD. On the other hand, qPCR is more advantageous than nested-PCR for processing large amounts of samples since it does not need post processing steps for visualization of results and the level of bacterial infection can be quantified. Therefore by improving the diagnosis of RSD in field plants, it can be applied to monitor losses caused by the disease, as a tool to monitor the efficacy of sanitation procedures and in the development of new sanitizers for Lxx.