Abstract: The present invention provides methods and compositions for determining the presence and/or amount of pathogenic Leptospira in a test sample. In particular, substantially purified oligonucleotide primers and probes are described that can be used for qualitatively and quantitatively detecting pathogenic Leptospira nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for assessing internal quality control of the Leptospira assay.