To date, the genomes of several mycoplasmas have been cloned in Saccharomyces cerevisiae cells, including Mycoplasma genitalium, M. pneumoniae, and M. mycoides. In this work, the 1-Mb genome of Mycoplasma gallisepticum S6 was cloned in S. cerevisiae. A transposon vector was constructed to deliver a synthetic S. cerevisiae replicon and a selective marker into M. gallisepticum genome. Transplantation of the M. gallisepticum genome with the integrated yeast replicon to yeast cells was performed using two approaches, polyethylene glycol (PEG)-induced transformation of yeast spheroplasts with intact genomic DNA isolated from M. gallisepticum cells and prepared in aragose plugs and PEG-induced fusion of M. gallisepticum cells with yeast spheroplasts. Both of the approaches yielded S. cerevisiae strains with the cloned M. gallisepticum genome, as was verified by nanopore sequencing.