The genus Leptospira is composed of spirochetes that are morphologically indistinguishable and cannot be differentiated by phenotypic methods. Determining the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Pulsed-field gel electrophoresis (PFGE) of Leptospira has had limited use in few laboratories. In this study, we modified the existing PFGE protocol to reduce time to completion and developed a PFGE database using the restriction endonuclease Not I to generate PFGE profiles for the identification and evaluation of Leptospira. Reference strains (n = 206) from a collection maintained by the Centers for Disease Control and Prevention were studied. Eighty-nine percent of the serovars produced distinct patterns, and different strains of the same serovar showed profiles that were highly similar. Pulsed-field gel electrophoresis of Leptospira is a useful tool for molecular characterization of reference serovars, the investigation of potentially new species or serovars, and ultimately for the routine identification of clinical isolates.