Three infectious bronchitis virus (IBV) strains, isolated from suspected Tunisian broiler flocks, were characterized as variant viruses using genotyping and serotyping techniques. They were compared with commonly used vaccine strains, including 793/B, D274, and Massachusetts types. Reverse transcription-PCR-restriction fragment length polymorphism, nucleotide sequencing, and GenBank BLAST database analyses of the hypervariable region of the S1 subunit of the virus spike gene showed that the three isolates, designated TN20/00, TN200/01, and TN335/01, share from 64% to 82% homologies between each other but are very different from the H120 strain, the only infectious bronchitis vaccine used in Tunisia. In addition, they showed from 57% to 78% similarities with the European genotypes, including D274 and 793/B. Phylogenetic data allowed classification of the three Tunisian isolates as new genotypes placed inside the same genetic group as the CR88121 and D274 genotypes but very distant from the Massachusetts genotype. Cross-virus neutralization tests confirmed the genotyping results and showed that both TN200/01 and TN335/01 isolates are serologically related, whereas the TN20/00 is closer to TN335/01 than to TN200/01. Moreover, all three Tunisian isolates are closely related to the European variant serotypes, including the CR88121 and the D274 strains, but none is serologically related to the H120 vaccine strain. These data demonstrated, for the first time in Tunisia, the cocirculation of IBV variant serotypes along with the Massachusetts type, causing severe clinical diseases and high economic losses to the poultry industry.