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Allotment gardens are a key feature of the urban landscape, providing numerous benefits beyond those of food production. These include ecosystem services such as local climate modification, pollination, and providing a pleasant location for socialising. The biodiversity of flora underpins many of these ecosystem services. The aims of this study are to assess the spontaneous vascular flora of urban allotment gardens and derive estimates of the species richness and diversity. In doing so, the specific position of allotment gardens within urban green infrastructure will be revealed and the role of allotment users in shaping local biodiversity determined. 358 species of spontaneous flora were recorded in 11 representative allotment garden estates (total area 150 ha) in Poznan, Poland. Fourteen biodiversity features were taken into account. The investigated areas exhibited high plant species richness, high diversity, low synanthropisation level, and contained valuable geobotanical elements, including plants of key importance for the European Community. For these reasons, allotment gardens should be considered as biodiversity hotspots for native species within urban green infrastructure. Based on an analysis of the spatial and functional organization of 110 individual plots, four types of usage, and corresponding plant diversity have been distinguished. The composition of the flora depends mostly on spatial and functional organization of allotment gardens and, to a much lesser extent, on the habitat conditions, which on individual plots varied very little. The high species richness and diversity in allotment gardens has important consequences for urban planning.