This work is devoted to the study of the correlation between mutational variability and cytotoxic properties of the oncolytic Sendai virus when using different culture systems. We have shown that, as a result of 12 consecutive passages in the allantoic fluid of chick embryos, Sendai virus accumulates in it in high titers without changing the RNA genomic sequence and cytotoxic properties in relation to melanoma (Mel8) and human glioma cells (U87MG). However, for the drugs obtained in this system, there is a problem of allergenicity and standardization. The solution could be an adaptation of the virus to cell culture. We used cell cultures 4647 (African green monkey kidney cells) and 293 (human embryonic kidney cells) that are certified for vaccine production in Russia. It turned out that in the course of the reproduction of the virus in these cell cultures during 21–25 passages, accumulation of mutations with the highest density of nonsynonymous substitutions in the F and HN genes, which encode the surface proteins of the virion and ensure its infectivity, takes place. The accumulation of mutations leads to a significant decrease in the cytotoxic activity of Moscow strain of Sendai virus in respect of melanoma and human glioma cells. Reverse passaging on chick embryos of the Sendai virus, adapted to cells 4647, provides partial elimination of adaptive mutations and restoration of oncolytic activity of the virus. Mutations acquired during adaptation to cells 293 are not eliminated in the process of tenfold reverse passaging in chick embryos and the oncolytic activity of the virus does not recover. Thus, the stability of the Sendai virus genome is important for the implementation of its oncolytic potential, and this must be taken into account when choosing a technique for the production of antitumor drugs based on this virus.