This article describes a lethal case of leptospirosis that occurred in Southern Russia. TheLeptospirastrain was isolated and characterized using a microscopic agglutination test, MALDI-TOF mass spectrometry, targeted PCR, and high-throughput sequencing. We show that molecular and mass-spectrometry methods can be an alternative to conventional methods of leptospirosis diagnostics andLeptospirastudy, which require highly qualified staff and can be performed only at specialized laboratories. We also report the first whole genome ofL.interrogansisolated in Russia.