The Wyeth strain of vaccinia virus (VACV) produced by Wyeth Pharmaceuticals was supposedly used to manufacture the old freeze-dried American smallpox vaccine, Dryvax, until its discontinuation in 2008. Although the genomic sequences of numerous Dryvax clones have been reported, data on VACV-Wyeth genomes are still lacking. Genomic analysis of old VACV strains is relevant to understand the evolutionary relationships of smallpox vaccines, particularly with the recent resumption of smallpox vaccination in certain population groups as an attempt to control the worldwide monkeypox outbreak. Here we analyzed the complete genome sequences of three VACV-Wyeth clonal isolates obtained from a single seed vial donated to the Brazilian eradication program in the 1970s. Wyeth clones show >99.3% similarity to each other and >95.3% similarity with Dryvax clones, mapping together in clade I of the vaccinia group. Although the patterns of SNPs and INDELs comparing Dryvax and Wyeth clones are overall uniform, important differences were detected particularly at the ends of the genome. In addition, we detected recombinant events of clone Wyeth A111 and the Dryvax clone Acam2000, suggesting that other regions of the genomes may have similar patchy patterns of recombination. A small-scale serological survey using VACV-Wyeth as antigen in ELISA assays revealed that 63 of the 65 individuals born before the end of smallpox vaccination in Brazil still have anti-VACV IgG antibodies, demonstrating the usefulness of the VACV-Wyeth strain in future extended serological studies of the Brazilian population.