The mammalian CNS is composed of an extremely complex meshwork of highly ordered interconnections among billions of neurons. To understand the diverse functions of this neuronal network we need to differentiate between functionally related and nonrelated elements. A powerful labeling method for defining intricate neural circuits is based on the utilization of neurotropic herpesviruses, including pseudorabies virus and herpes simplex virus type 1. The recent development of genetically engineered tracing viruses can open the way toward the conception of novel tract-tracing paradigms. These new-generation tracing viruses may facilitate the clarification of problems, which were inaccessible to earlier approaches. This article first presents a concise review of the general aspects of neuroanatomical tracing protocols. Subsequently, it discusses the molecular biology of alpha-herpesviruses, and the genetic manipulation and gene expression techniques that are utilized for the construction of virus-based tracers. Finally, it describes the current utilization of genetically modified herpesviruses for circuit analysis, and the future directions in their potential applications.