Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.