The prevalence of leptospirosis is generally high in domestic animals and rodents in Tanzania. Identification of Leptospira isolates from cattle was carried out to establish prevalent Leptospira serovars. Serological typing was done based on monoclonal antibodies and the standard cross-agglutination absorption test. Molecular typing involved pathogenic- and saprophytic-specific PCRs and a PCR specifically amplifying DNA from the species Leptospira kirschneri. DNA fingerprinting with primers derived from sequences of insertion elements IS1500 and IS1533 was carried out. Both serological and molecular characterization indicated that one of the Leptospira isolates, coded RM1, represents a new serovar of the species L. kirschneri of serogroup Icterohaemorrhagiae. The serovar name Sokoine is proposed for this new Leptospira isolate.