Rabies is a neglected zoonotic disease with veterinary and public health significance, particularly in Africa and Asia. The current knowledge of the epidemiology of rabies in Mozambique is limited because of inadequate sample submission, constrained diagnostic capabilities and a lack of molecular epidemiological research. We wanted to consider the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) for rabies diagnosis at the diagnostic laboratory of the Central Veterinary Laboratory (CVL), Directorate of Animal Science, Maputo, Mozambique. Towards this aim, as a training exercise at the World Organisation for Animal Health (OIE) Rabies Reference Laboratory in South Africa, we performed the DRIT on 29 rabies samples from across Mozambique. With the use of the DRIT, we found 15 of the 29 samples (52%) to be negative. The DRIT-negative samples were retested by DFA at the OIE Rabies Reference Laboratory, as well as with an established real-time Polymerase chain reaction, confirming the DRIT-negative results. The DRIT-positive results (14/29) were retested with the DFA and subsequently amplified, sequenced and subjected to phylogenetic analyses, confirming the presence of rabies RNA. Molecular epidemiological analyses that included viruses from neighbouring countries suggested that rabies cycles within Mozambique might be implicated in multiple instances of cross-border transmission. In this regard, our study has provided new insights that should be helpful in informing the next steps required to better diagnose, control and hopefully eliminate rabies in Mozambique.