A novel glyphosatetolerant EPSPS gene AroAI.va* derived from Isoptericola variabilisn and driven by maizederived composite ciselement was efficiently and simultaneously expressed in the plastids of leaves and nongreen tissues of tobacco. The results of transcription and proteinlevel determination of the homoplastomic transplastomic lines showed that the AroAI.va* gene was effectively expressed in the leaf and root tissues. The T0 seeds of homoplastomic transplastomic plants pLSZ3#2 and pLSZ3#3 tolerated more than 12 mmol/L glyphosate treatment. The transgenic plants cultivated in the greenhouse tolerated the spraying treatment with glyphosate at a dose of 1 800 mg/L. Reciprocal crosses between wildtype and transgenic plants confirmed that the glyphosateresistant transgenic plants obtained by plastid transformation had the characteristics of maternal inheritance.