Genetic and molecular methods to investigate the pathogenesis of the poultry respiratory pathogen Mycoplasma gallisepticum are quite limited. Therefore, the objective of this study was to design and evaluate a functional genomics approach to identify M. gallisepticum genes involved in colonization of the poultry respiratory tract. To serve as a transcriptional reporter, a promoterless lacZ gene from Escherichia coli was cloned into the Tn4001 transposon. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain, and a bank of 1386 transposon mutants containing lacZ fusions to mycoplasma chromosomal DNA was assembled. Each mycoplasma clone containing the lacZ reporter was independently screened in the chicken tracheal ring organ culture (TROC) model system for increased production of β-galactosidase. A twofold or greater increase in β-galactosidase was consistently observed for eight mutants. In one of the mutants, the transposon was inserted in a pMGA gene encoding a cell surface adhesin involved in hemagglutination. Therefore, these data indicate that screening of a M. gallisepticum transposon reporter bank with a chicken TROC model is useful for the identification of genes induced during poultry colonization and virulence.