As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (d S/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype. © 2006 Houghton Trust Ltd.