Leptospirawas investigated in kidneys (n= 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolateLeptospira, followed by theLipL32qPCR to detectLeptospiraDNA. TheSecYgene region was amplified, sequenced, and analyzed forLipL32qPCR-positive samples orLeptospiraisolates. The overall frequency of isolation ofLeptospiraspp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (p> 0.05). However, withLipL32qPCR, the overall frequency ofLeptospiraDNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (p= 0.03). Based on 22SecYsequences, the phylogenetic tree identified theL. interroganscluster with serovar Icterohaemorrhagiae and theL. borgpeterseniicluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization ofLeptospiraspp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of whichL. borgpeterseniiserovar Hardjo bovis is not part. Our data show that pathogenicL. interrogansandL. borgpeterseniiare circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.