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RESUMO.[Diagnóstico de leptospirose canina: avaliação de dois ensaios de PCR em comparação com o teste de microaglutinação.] O diagnóstico definitivo da leptospirose canina é geralmente realizado demonstrando a seroconversão em amostras do paciente no período agudo e de convalescença por serologia. No entanto, a aplicação de técnicas de PCR pode contribuir para a confirmação de casos suspeitos num período de tempo mais curto. O objetivo deste estudo foi avaliar dois ensaios de PCR publicados em humanos (PCR‐lipL32 em tempo real e PCR‐rrs convencional) em culturas puras e em amostras de sangue com anticoagulante, soro e urina experimentalmente contaminados. Posteriormente, investigamos a utilidade de ambos os ensaios de PCR em amostras clínicas de cães com suspeita de leptospirose tomando a técnica de microaglutinação (MAT) como referência. A sensibilidade analítica foi de 1 e 10 genoma equivalente por reação para PCR‐lipL32 em tempo real e para PCR‐rrs convencional, respectivamente. Ambos os ensaios amplificaram ABSTRACT.Martin P.L., Stanchi N.O., Brihuega B.F., Bonzo E., Galli L. & Arauz M.S. 2019. Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test. Pesquisa Veterinária Brasileira 39(4):255-262. Servicio Central de Laboratorio, Hospital Escuela, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Av. 60 y 118, CC 296, La Plata, CP 1900, Buenos Aires, Argentina. E‐mail: plmartin@fcv.unl.edu.ar Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real‐time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real‐time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real‐time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. The lipL32 real‐time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.