The Amazon River watershed and its associated plume comprise a vast continental and oceanic area. The microbial activities along this continuum contrib- ute substantially to global carbon and nutrient cycling, and yet there is a dearth of information on the diversity, abundance, and possible roles of viruses in this globally important river. The aim of this study was to elucidate the diversity and structure of virus assemblages of the Amazon River-ocean continuum. Environmental viral DNA sequences were obtained for 12 locations along the river’s lower reach (n 5) and plume (n 7). Sequence assembly yielded 29,358 scaffolds, encoding 82,546 viral proteins, with 15 new complete viral genomes. Despite the spatial connectivity me- diated by the river, virome analyses and physical-chemical water parameters clearly distinguished river and plume ecosystems. Bacteriophages were ubiquitous in the continuum and were more abundant in the transition region. Eukaryotic viruses oc- curred mostly in the river, while the plume had more viruses of autotrophic organ- isms (Prochlorococcus, Synechococcus) and heterotrophic bacteria (Pelagibacter). The viral families Microviridae and Myoviridae were the most abundant and occurred throughout the continuum. The major functions of the genes in the continuum in- volved viral structures and life cycles, and viruses from plume locations and Tapajós River showed the highest levels of functional diversity. The distribution patterns of the viral assemblages were defined not only by the occurrence of possible hosts but also by water physical and chemical parameters, especially salinity. The findings pre- sented here help to improve understanding of the possible roles of viruses in the organic matter cycle along the river-ocean continuum.