We compared three cold-adapted live attenuated influenza vaccine strains prepared by reverse genetics methods on the basis of master donor virus A/Leningrad/134/17/57 and influenza H7N9 strains A/Anhui/1/2013 and A/Shanghai/1/2013. Two strains based on A/Anhui/1/2013 differed by amino acid positions 123 and 149 in HA1 (123N/149N; 123D/149D). All strains efficiently replicated in developing chicken embryos; A/Shanghai/1/2013-based strain and A/Anhui/1/2013-123N/149N variant were characterized by reduced replication in MDCK cells. Strains based on A/Anhui/1/2013 virus agglutinated erythrocytes with α2,3- and α2,6-linked sialic acid residues, whereas strain A/Shanghai/1/2013 only α2,3. In experiments with BALB/c mice, Anhui-123D/149D strain was most immunogenic and induced high crossreactive humoral immune response, therefore it can be recommended as the model virus for the construction of recombinant vector vaccines based on live attenuated influenza vaccine.