Smallpox was eradicated using variant forms of vaccinia virus-based vaccines. One of these was Dryvax, a calf lymph vaccine derived from the New York City Board of Health strain. We used genome-sequencing technology to examine the genetic diversity of the population of viruses present in a sample of Dryvax. These studies show that the conserved cores of these viruses exhibit a lower level of sequence variation than do the telomeres. However, even though the ends of orthopoxviruses are more genetically plastic than the cores, there are still many telomeric genes that are conserved as intact open reading frames in the 11 genomes that we, and 4 genomes that others, have sequenced. Most of these genes likely modulate inflammation. Our sequencing also detected an evolving pattern of mutation, with some genes being highly fragmented by randomly assorting mutations (e.g., M1L), while other genes are intact in most viruses but have been disrupted in individual strains (e.g., I4L in strain DPP17). Over 85% of insertion and deletion mutations are associated with repeats, and a rare new isolate bearing a large deletion in the right telomere was identified. All of these strains cluster in dendrograms consistent with their origin but which also surprisingly incorporate horsepox virus. However, these viruses also exhibit a "patchy" pattern of polymorphic sites characteristic of recombinants. There is more genetic diversity detected within a vial of Dryvax than between variola virus major and minor strains, and our study highlights how propagation methods affect the genetics of orthopoxvirus populations.