Leptospirosis is a zoonotic infection for which diagnosis is difficult. It has appeared as a global emerging infectious disease over recent years. Genotype determination often requires a Leptospira strain obtained by culture, which is a long and fastidious technique. A method based on multilocus variable number tandem repeat analysis (MLVA) to determine the genotype of Leptospira interrogans, performed directly on blood or urine samples, is proposed. This method was applied to a fatal case of leptospirosis for which the geographical origin of infection was unknown. This technique will allow a genotype to be obtained for L. interrogans, even when cultures remain negative.