We examined spike (S) gene sequences of the virus populations of four different commercial ArkDPI-derived infectious bronchitis coronavirus vaccines before and during a single passage in specific pathogen free chickens. We found different degrees of genetic heterogeneity among the four vaccines before passage in chickens, ranging from no apparent heterogeneity to heterogeneity in 20 positions in the S gene. In all except one position, nucleotide differences were non-synonymous. The majority of amino acid differences were in the S1 subunit of the protein. For three of the four ArkDPI-derived vaccines, a single subpopulation with an S gene sequence distinct from the vaccine majority consensus at 5 to 11 codons was selected in chickens within 3 days after ocular vaccination. In contrast, we obtained no evidence for selection of specific subpopulations of the fourth ArkDPI-derived vaccine or Massachusetts or DE072 serotype vaccines. The virus subpopulations within each vaccine selected by chickens are similar in their S1 gene sequences, but distinct in the 3' portion of the S2 subunit gene for each of the three vaccines. In the S1 gene, the selected subpopulations are more similar to the virulent parental ArkDPI isolate than to the predominant vaccine population. The different proportions of distinct subpopulations in Ark vaccines apparently more fit for replication in the respiratory tract of chickens might cause different degrees of damage to respiratory epithelium and/or immune responses in vaccinated chickens. Sequence comparisons provided no evidence to support that ArkDPI-like field isolates were derived directly from host-selected vaccine virus subpopulations.