Rabies are one of the major zoonoses in Mali. The control of this disease is based on the use of effective diagnostic techniques, the management of human cases after exposure and medical prophylaxis of animals. Diagnosis is usually made by detection of viral antigen by direct immunofluorescence (IF). Due to the increasing variability of the genus Lyssavirus observed, it was found necessary to use a complementary technique, namely reverse transcription in polymerase chain reaction (RT-PCR). The objective of the present study was to assess the diagnostic performance of IFD and RT-PCR for the diagnosis of animal rabies in Mali with a view to characterizing the different strains identified at the molecular level (gene of rabies virus nucleoprotein N). Thus, the analysis of forty (40) brain samples from dogs suspected of having rabies, revealed 36 positive cases for IFD, i.e. 90% and 33 for RT-PCR, i.e. 82.5.% and a concordance rate of 82.5%. Analysis of the segment of the gene of nucleoprotein (N) showed that the rabies viruses circulating in Mali belong to the Africa 2 lineage (Africa 2). Statistical analyzes showed the existence of a significant difference (P=0.001<0.005) between the performance of the two techniques. The present study shows the usefulness of the complementarity of the two tests as an alternative for confirming the diagnosis of rabies.