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Rabies is a lethal zoonotic encephalomyelitis that causes an estimated 59,000 human deaths yearly worldwide. Although developing countries of Asia and Africa bear the heaviest burden, surveillance and disease detection in these countries is often hampered by the absence of local laboratories able to diagnose rabies and/or the difficulties of sample shipment from low-access areas to national reference laboratories. Filter papers offer a convenient cost-effective alternative for the sampling, shipment, and storage of biological materials for the diagnosis of many pathogens including rabies virus, yet the properties of diagnostic tests using this support have not been evaluated thoroughly. Sensitivity and specificity of molecular diagnosis of rabies infection using a reverse transcription followed by a hemi-nested polymerase chain reaction (RT-hn-PCR) either directly on brain tissue or using brain tissue dried on filter paper were assessed on 113 suspected field animal samples in comparison to the direct fluorescent antibody test (FAT) recommended by the World Health Organization as one of the reference tests for rabies diagnosis. Impact of the duration of the storage was also evaluated. The sensitivity and the specificity of RT-hn-PCR i) on brain tissue were 96.6% (95% CI: [88.1–99.6]) and 92.7% (95% CI: [82.4–98.0]) respectively and ii) on brain tissue dried on filter paper 100% (95% CI: [93.8–100.0]) and 90.9% (95% CI: [80.0–97.0]) respectively. No loss of sensitivity of RT-hn-PCR on samples of brain tissue dried on filter paper left 7 days at ambient temperature was detected indicating that this method would enable analyzing impregnated filter papers sent to the national reference laboratory at ambient temperature within a 1-week shipment time. It could therefore be an effective alternative to facilitate storage and shipment of samples from low-access areas to enhance and expand rabies surveillance., Author summary Dogs are responsible for 99% of human rabies deaths. Facilitating diagnostic of rabid dogs can help further identify i) people who were exposed to rabies in order to encourage them to seek post exposure prophylaxis and ii) other exposed animals to break the transmission chain (i.e. prevent them from further transmitting the virus to other people and animals). Yet, the reference diagnostic method for animal rabies requires brain samples to be collected post-mortem and shipped under temperature-controlled conditions. The shipping of such samples is complex in low-access areas, especially in low income countries, which are often those that bear the heaviest rabies burden. Filter papers offer a convenient alternative for biological sample shipment and storage. Here the efficiency of a molecular diagnostic method applied to brain tissue dried on filter paper is compared to one of the reference methods, the direct fluorescent antibody test. Our results show it has an excellent sensitivity (it does not miss any positive samples), even when filter papers are left 7 days at ambient temperature. These results let us foresee a cost-effective alternative facilitating shipment, storage and testing of samples from rabies suspected animals from low-access areas. This could considerably enhance and expand rabies surveillance in low-income countries, allowing a more comprehensive evaluation of rabies burden, and thus reinforcing arguments for allocating funds to rabies control policies.