Leaf scald is an important disease of sugarcane with erratic symptom expression. Latency represents a threat to germplasm exchange, and erratic symptom development makes accurate evaluation of disease resistance during breeding and selection problematic. Real-time quantitative polymerase chain reaction (qPCR) assays for Xanthomonas albilineans, the causal agent of leaf scald, were developed and evaluated for the sensitive, specific detection and quantification of the pathogen. Assays with SYBR Green primers and TaqMan probe and primers derived from the albicidin toxin biosynthesis gene cluster efficiently and reproducibly amplified X. albilineans. Detection was more sensitive with qPCR compared with conventional PCR. Assays were specific for X. albilineans and sap extracts did not inhibit the qPCR reaction. Leaf-scald-resistant and -susceptible cultivars were distinguished by infection incidence, disease severity, and X. albilineans population determined by SYBR Green qPCR in both greenhouse and field experiments. Populations of X. albilineans varied in different tissues. Differences were the greatest within tissues in resistant cultivars, and bacterial populations in systemically infected, young, not yet fully emerged leaves exhibited the greatest differences between resistant and susceptible cultivars. The results demonstrate that qPCR is a highly sensitive method for the detection of X. albilineans that could provide a reliable method for leaf scald resistance screening.