Since 1980 there has been global eradication of smallpox due to the success of the vaccination programme using vaccinia virus (VACV). During the eradication period, distinct VACV strains circulated, the Lister strain being the most commonly employed in Europe. Analysis of the safety of smallpox vaccines has suggested that they display significant heterogeneity. To gain a more detailed understanding of the diversity of VACV strains it is important to determine their genomic sequences. Although the sequences of three isolates of the Japanese Lister original strain (VACV-LO) are available, no analysis of the relationship of any Lister sequence compared to other VACV genomes has been reported. Here, we describe the sequence of a representative clonal isolate of the Lister vaccine (VACV-List) used to inoculate the French population. The coding capacity of VACV-List was compared to other VACV strains. The 201 open reading frames (ORFs) were annotated in the VACV-List genome based on protein size, genomic localization and prior characterization of many ORFs. Eleven ORFs were recognized as pseudogenes as they were truncated or fragmented counterparts of larger ORFs in other orthopoxviruses (OPVs). The VACV-List genome also contains several ORFs that have not been annotated in other VACVs but were found in other OPVs. VACV-List and VACV-LO displayed a high level of nucleotide sequence similarity. Compared to the Copenhagen strain of VACV, the VACV-List sequence diverged in three main regions, one of them corresponding to a substitution in VACV-List with coxpox virus GRI-90 strain ORFs, suggestive of prior genetic exchanges. These studies highlight the heterogeneity between VACV strains and provide a basis to better understand differences in safety and efficacy of smallpox vaccines.