Cyprinid herpesvirus 3 (CyHV-3) is the archetypal fish alloherpesvirus and the etiologic agent of a lethal disease in common and koi carp. To date, the genome sequences of only four CyHV-3 isolates have been published, but no comparisons of the biologic properties of these strains have been reported. We have sequenced the genomes of a further seven strains from various geographical sources, and have compared their growth in vitro and virulence in vivo. The major findings were: (i) the existence of the two genetic lineages previously described as European and Asian was confirmed, but inconsistencies between the geographic origin and genotype of some strains were revealed; (ii) potential inter-lineage recombination was detected in one strain, which also suggested the existence of a third, as yet unidentified lineage; (iii) analysis of genetic disruptions led to the identification of non-essential genes and their potential role in virulence; (iv) comparison of the in vitro and in vivo properties of strains belonging to the two lineages revealed that inter-lineage polymorphisms do not contribute to the differences in viral fitness observed; and (v) a negative correlation was observed among strains between viral growth in vitro and virulence in vivo. This study illustrates the importance of coupling genomic and biologic comparisons of viral strains in order to enhance understanding of viral evolution and pathogenesis.