Arboviruses (Arthropod-borne viruses) compose a large group of zoonotic viruses that have complex cycles and often cause diseases in humans. This study was aimed at performing a systematic review of molecular diagnostic methods for the detection of arboviruses in clinical samples from Brazil to emphasize the importance of a differential diagnosis. Articles on arbovirus diagnostic methods were searched in databases using descriptors and selection criteria. A total of 19 articles were found that described techniques that may be used for the differential diagnosis of arboviruses. RT-PCR, nested RT-PCR and real-time RT-PCR were the main methods. The samples were collected from the Brazilian Amazon and the state of São Paulo for the diagnosis of dengue, Oropouche fever, yellow fever, Saint Louis encephalitis and Mayaro virus disease. Classical diagnostic methods were rarely used. In addition, molecular methods are not yet fully standardized because several methods did not detect arboviruses. Furthermore, a diagnosis that is based only on clinical and epidemiological data would be premature. Therefore, new entomological research and new differential molecular methods should be performed for the possible isolation of these unknown viruses to contribute to the diagnosis of arboviruses. Thus, additional research should be conducted because these diseases are emerging and reemerging in several countries.