Mycoplasma gallisepticum is an important respiratory pathogen of commercial poultry causing considerable economic losses worldwide. Because there are relatively few genetic tools available for manipulation of M. gallisepticum, and the mycoplasmas in general, we have limited understanding of gene function in these pathogens. M. gallisepticum strain S6 possesses an endogenous CRISPR/Cas system that may be able to be adapted for use in genome engineering in this species. To generate guide RNA precursors to direct the Cas proteins in M. gallisepticum, two CRISPR arrays were assembled: One with three Direct Repeats interspersed with two spacers, and the other with two Direct Repeats surrounding one spacer. Each CRISPR array was placed under the control of the vlhA1.1 promoter in a plasmid carrying the oriC of M. imitans (pCRISPR and pCRISPR_1sp). The spacers were designed to target regions on the ksgA gene, which encodes a 16S rRNA adenine dimethyl transferase. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside kasugamycin. Electrocompetent M. gallisepticum strain S6 cells were transformed with pCRISPR and pCRISPR_1sp, and cultured on mycoplasma agar plates containing concentrations of kasugamycin above the MIC. PCR assays targeting the ksgA gene of the phenotypically resistant colonies transformed with pCRISPR indicated some polymorphism within the targeted region. Sequence analysis of the ksgA gene in five of those colonies suggested that M. gallisepticum may utilize a nonhomologous end joining repair system, which results in deletion or duplication of a short DNA segment at double-stranded breaks in the genome. The efficiency of endogenous CRISPR/Cas system modification using one or two guide RNAs is currently being evaluated by sequencing and examining the growth rate of populations of M. gallisepticum transformed with pCRISPR or pCRISPR_1sp in presence of kasugamycin. This study may enhance our capacity to genetically modify this important pathogen.