Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia virus with restricted replication in human cells. The virus serves as an ideal vaccine vector suitable for safe use even in immune-compromised individuals. With its inherently large packaging capacity, expression cassettes encoding bulky genes can be inserted into deletion regions within the MVA genome. These deletion sites develop during the process of the attenuation of the virus by passage in Chicken Embryo Fibroblasts (pCEFs). Transgene stability in MVA is important to assure immunogenicity and efficacy. In the present study, we assessed the effect of substantial passage of recombinant MVA vectors on the stability of expression cassette encoding SIV Gag/Tat genes inserted at the Del-II site, as part of generating a vaccine to protect from HIV. Our data indicated that after 15 passages there was a significant loss or mutation of the inserted genes.