Epstein-Barr virus (EBV) infects most of the world’s population and is causally associated with several human cancers, but little
is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published
wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have
sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the
first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all
strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced
from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined
almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism
(SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some
T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection,
both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination
between strains, which provides a further mechanism for the generation of diversity. Our results provide the first
global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further
understand the genetics of EBV infection.