Background: First-line biological diagnosis of malaria in Mali is based on the use of rapid diagnostic tests (RDT), which detect in the blood the Histidin Rich Protein 2 antigen, specific to Plasmodium falciparum. Our study, based on a real-time polymerase chain reaction (qPCR) gold-standard diagnosis, aims to evaluate the performance of RDTs used in Mali and to describe the distribution of Plasmodium species in each administrative regions of Mali; Methods: We collected RDT cassettes in 47 sites of the nine regions of Mali. We randomly selected 150 malaria-negative and up to 30 malaria-positive RDTs from each regions. DNA was extracted from the RDTs’ nitrocellulose strip and then assayed with a pan-Plasmodium qPCR. Positive samples were then analyzed with qPCRs specific for P. falciparum, P. malariae, P. vivax, and P. ovale; Results: Of the 1,389 tested by qPCR RDTs, 258 (18.6%) were positive for Plasmodium spp. P.falciparum was the most predominant (97.3%). However, the prevalence of P.vivax reach 21.1% in the Ménaka region in the north; Conclusion: Overall, RDT diagnostic indices are adequate for the biological diagnosis of malaria in Mali. Our finding support the adjustment of RDTs to the local epidemiology.