Viral emergence can result from the adaptation of endemic pathogens to new or altered host environments, a process that is strongly influenced by the underlying sequence diversity. To determine the extent and structure of intrahost genetic diversity in a recently emerged single-stranded DNA virus, we analyzed viral population structures during natural infections of animals with canine parvovirus (CPV) or its ancestor, feline panleukopenia virus (FPV). We compared infections that occurred shortly after CPV emerged with more recent infections and examined the population structure of CPV after experimental cross-species transmission to cats. Infections with CPV and FPV showed limited genetic diversity regardless of the analyzed host tissue or year of isolation. Coinfections with genetically distinct viral strains were detected in some cases, and rearranged genomes were seen in both FPV and CPV. The sporadic presence of some sequences with multiple mutations suggested the occurrence of either particularly error-prone viral replication or coinfection by more distantly related strains. Finally, some potentially organ-specific host effects were seen during experimental cross-species transmission, with many of the mutations located in the nonstructural protein NS2. These included residues with evidence of positive selection at the population level, which is compatible with a role of this protein in host adaptation.Emerging viruses that gain new host ranges are major threats to human and animal health. However, the evolutionary mechanisms that allow viruses to infect new hosts and establish self-sustaining transmission chains are complex and far from understood. In most cases studied to date, several mutations together determine the new host range, but where, when, and how these mutations arise have remained largely enigmatic. Many cases of viral emergence probably represent multistage adaptations to an altered host environment, where the initial emerging virus is poorly adapted to the recipient host and causes only inefficient transmission. Further mutations are then required for complete adaptation to the new host. The success of the new pathogen is therefore likely to be highly influenced by the genetic diversity of the viral population, with more variable viral populations being particularly prone to generating successful emerging infections.The extent of genetic diversity present within viral populations is determined largely by a balance between erroneous replication (measured as the mutation rate) and purifying selection (reflected in the population substitution rate, defined as the number of fixed mutations/nucleotide site/year), with particularly high error rates in RNA viruses that replicate using RNA-dependent RNA polymerases (15), or, in the case of retroviruses, reverse transcriptases. Although large doublestranded DNA viruses possess mutation rates far lower than those seen in RNA viruses, some small single-stranded DNA