Viral retrograde transneuronal labelling has become an important neuroanatomical tract-tracing tool for characterization of limbic neuronal networks. Recently, dual viral retrograde transneuronal labelling has been introduced; a method employing differential transgene expression of two genetically engineered virus strains to identify double infected cells with selective antibodies. In this way, interactions of parallel networks can be revealed. The use of this method will increase the understanding of the function of the limbic system, for example in the maintenance of metabolic homeostasis, but is associated with limitations related to the use of genetically engineered virus strains. Virulence, speed of replication and retrograde transport may be affected by the insertion or deletion of genes in the viral genome. Moreover, the rate of replication and transport can be affected by the immune system of the host and competition between the two viruses. There may be selective affinity of the virus strain for the sympathetic or parasympathetic systems. False negative results are the most important risk in dual viral labelling addressed in this review. Several control experiments are presented that can help to reduce the risk of obtaining false negative results.