In many areas of the agriculturalized Pampas of Argentina, rural roadsides (RRS) are the only relics of the grassland biome that originally dominated the region. Nowadays, no policies regulate the use and preservation of these environments. We assessed plant species diversity in 28 RRS in a representative area of the Southern Pampean region, and investigated the degree to which some RRS variables (width, slope, type, and density of RRS in a circular area of 2000 m in diameter) explain their floristic assemblages and species richness. We listed 107 species belonging to 25 families. The most species-rich families were Poaceae (33 species) and Asteraceae (22 species). Fifty percent of the species were native to the region and 58% perennial. There was a predominance of herbaceous dicots (68%), and 12 species (11%) were endemic to the region. A canonical correspondence analysis showed that the density of neighboring RRS significantly explained plant species assemblages. Exotic species richness decreased with the width of the RRS. We expect that this information and proper management will promote the potential of RRS for nature conservation as we consider RRS important refuge for grassland plants.