Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.