Recombination between co-infecting herpesvirus DNA genomes is an important process affecting viral population diversity and evolution. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when co-infecting viral genomes interact. To study the spatiotemporal requirements for inter-genomic recombination, we developed an assay with dual-color fluorescence in situ hybridization enabling detection of homologous recombination (HR) between different co-infecting HSV-1 pairs. Our results revealed that when viral RCs enlarge towards each other, there is detectable overlap of genomes from each virus. Infection with paired viruses that allow visualization of HR correlates with increased overlap of RCs. Taken together, these findings suggest that HR events take place during replication of HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce.