Pathogenic Leptospira, the causative agents of leptospirosis, comprise >200 serotypes (called serovars). Most have a restricted reservoir-host range, and some, e.g., serovar Copenhageni, are cosmopolitan and of public health importance owing to their propensity to produce severe, fatal disease in humans. Available serotyping approaches-such as multilocus sequence typing, core genome sequence typing, pulsed-field gel electrophoresis, and the cross-agglutination absorption test-are tedious and expensive, and require isolation of the organisms in culture media-a protracted and incredibly inefficient process-precluding their use in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. Here, we have developed a simple yet specific real-time qPCR assay-targeting a Leptospira-unique gene encoding a putative polysaccharide flippase-that provides intraspecies, serotype-defining (i.e., epidemiologically useful) information, and improves upon the sensitivity of preferred lipL32-based qPCR-based diagnostic tests. The assay, dubbed RAgI ("rage one"), is rapid and affordable, and reliably and specifically detects group I pathogenic Leptospira in culture, serum, and urine, with no detectable off-target amplification-even of the genetically related but low virulence group II pathogenic (formerly "intermediate") or nonpathogenic Leptospira. It retained 100% diagnostic specificity when tested against difficult sample types, including field-collected dog urine samples and environmental samples containing varied and complex microbial species-consortia. This assay holds considerable promise in the clinical setting, and for routine epidemiological and environmental surveillance studies. IMPORTANCE Leptospirosis is caused by a diverse group of pathogenic spirochetes comprising over 200 different serotypes. Some are widely reported and of public health importance owing to their propensity to produce severe, fatal disease in humans. Apart from their tedium and expense, current serotyping approaches require isolation of the organisms in culture media-a protracted and incredibly inefficient process-rendering them useless clinically and limiting their utilization in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. The 11108 qPCR-assay overcomes this barrier to progress via direct taxonomic and serotype classification of Leptospira from urine and serum samples, and hence, is the first qPCR-based prognostic test for human leptospirosis.