The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the gene locus () and the - intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and sites. Sequencing analysis implicated deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the - site, suggesting better accommodation of transgenes in this intergenic region. Thus, the - intergenic region may be more useful for transgene insertion for enhanced stability.