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This work provides an overview of plant diversity in the municipality of Rome (Italy) through an assessment of the flora in urban and suburban sectors of the city. It is aimed at providing the knowledge required to support proactive action for plant conservation. On the basis of a literature-derived catalogue and of an extensive survey campaign, the flora was investigated in terms of conservation interest; habitat types and locations that require protection measures were identified according to the occurrence of valuable native plants. Valuable species exclusive of the urban sector are threatened by the compaction trend affecting the urban fabric. Such species occur mainly in ruderal environments and fallows of archaeological sites and urban parks, near river courses and in remnants of natural forests. Valuable species exclusive of the suburban sector are threatened by urban sprawl. They occur prevalently in wet environments of the subcoastal strip and in coastal sands, Mediterranean maquis, tuffaceous gorges, sulphur springs and archaeological sites. The results highlight the need to preserve the complexity of the land mosaic, especially within the urban matrix, and to strengthen the existing environmental protection tools in the suburban area against foreseeable land cover changes.