The modified vaccinia virus Ankara (MVA), characterized by high immunogenicity and proven safety, is considered a promising vector for vaccine development. An undeniable advantage of the MVA vector is its high capacity and the ability to incorporate several transgenes into different loci of the viral genome, enabling the creation of multivalent vaccines that encode multiple antigens simultaneously. This study examined the expression of a transgene encoding influenza virus protein epitopes after its integration into five loci of the MVA genome. The results demonstrated that the level of transgene expression is independent of the integration locus. Additionally, the dynamics of the expression of a reporter gene encoding the Enhanced Green Fluorescent Protein (EGFP) were determined under the control of the vaccinia virus promoters p11, p13.5, pLEO160, p7.5, and mH5 upon insertion of the expression cassette into the F13L gene locus of MVA. The highest expression level, though with a delayed onset of protein synthesis, was achieved with the late promoter p11. Using the p13.5 promoter resulted in earlier synthesis of EGFP in cells and higher gene expression level compared to the promoters pLEO160, p7.5, and mH5, which provided similar levels and dynamics of the reporter gene expression. These findings may be useful for developing multi-antigenic MVA-vectored vaccines.