The process of avian influenza virus (AIV) evolution in a new host was investigated in the experiment in which ten serial passages of a turkey-derived H9N2 AIV were carried out in specific pathogen free chickens (3 birds/group) inoculated by oculonasal route. Oropharyngeal swabs collected 3 days post infection were used for inoculation of birds in the next passage and subjected to analysis using deep sequencing. In total, eight mutations in the consensus sequence were found in the viral pool derived from the 10th passage: four mutations (2 in PB1 and 2 in HA) were present in the inoculum as minority variants while the other four (2 in NP, 1 in PA and 1 in HA) emerged during the passages in chickens. The detected fluctuations in the genetic heterogeneity of viral pools from consecutive passages were most likely attributed to the selective bottleneck. The genes known for bearing molecular determinants of the AIV host specificity (HA, PB2, PB1, PA) contributed most to the overall virus diversity. In some cases, a fast selection of the novel variant was noticed. For example, the amino-acid substitution N337K in the haemagglutinin (HA) cleavage site region detected in the 6th passage as low frequency variant had undergone rapid selection and became predominant in the 7th passage. Interestingly, detection of identical mutation in the field H9N2 isolates 1-year apart suggests that this substitution might provide the virus with a selective advantage. However, the role of specific mutations and their influence on the virus adaptation or fitness are mostly unknown and require further investigations.