A survey of Leptospira spp. in wild mammals from the southern Pantanal of Mato Grosso do Sul was performed by microscopic agglutination test (MAT) and polymerase chain reaction (PCR). The serovars most frequently found were Hardjobovis (28%), Icterohemorhagiae (12%), M110/2006 strain (isolated from Cerdocyon thous, 16%), Canicola (L014 isolated from Bos Taurus, 4%), Whitcombi (4%), Pomona (20%), Autumnalis (12%) and Copenhageni (M9/99 isolated from Rattus norvegicus, 4%). From the 79 samples tested by PCR, 21 (26.58%) were positive, resulting in the amplification fragment of approximately 331pb. Two amplified fragments from C. thous were cloned, sequenced and identified as L. interrogans by phylogenetic analysis.