The objective of this study was to estimate the Trypanosoma evansi infection rate and epizootical status of wild and domestic animals from the Brazilian Pantanal region using a standardized polymerase chain reaction (PCR). We used a simple DNA extraction method based on Chelex resin (BioRad, USA) on blood eluted from filter paper confetti. Primers directed to repetitive nuclear DNA sequences were used in the PCR, and could detect 30 fg of T. evansi DNA. The analytical specificity of the assay was evaluated using T. evansi, T. rangeli, T. cruzi, Leishmania braziliensis, Crithidia fasciculata and Herpetomonas muscarum DNAs as templates and the technique showed the expected 164 bp specific band solely for Trypanozoon trypanosomes. The application of the standardized PCR protocol in 274 field samples from domestic and wild mammals from the Rio Negro (Brazilian Pantanal region), showed a general infection rate of 10.2% while the traditional parasitological technique (direct search of the protozoan by the microematocrit centrifugue technique) was able to determine infection in only 1.1% of the animals. The peccaries and feral pigs were found to be the animals most frequently infected with T. evansi (24.4% and 30.7%, respectively). Both sampling and extraction methods used herein, showed to be simple and efficient to be applied in epidemiological surveys using PCR.