The application of molecular approaches has revealed an immense biodiversity of lobose testate amoebae (Amoebozoa, Tubulinea, Elardia, Arcellinida). However, most newly discovered species are difficult or impossible to differentiate morphologically from already described forms, even though they are genetically distinct. Consequently, while diversity assessments based on sequence data overtake classical estimations of Arcellinida diversity, only a handful of morphologically divergent species have been discovered recently. These species are of key importance for reconstructing the evolutionary history of Arcellinida, including both morphology and morphogenesis. Here, we describe a large and conspicuous new species, Cylindrifflugia luciferina , found in an urban pond located in the Royal Botanical Garden of Madrid, Spain . We characterize it morphologically and molecularly, together with the closely related species Cylindrifflugia elegans . Our phylogenetic reconstruction based on the SSU rRNA gene brings evidence for three main well supported and morphologically consistent clades within the infraorder Cylindrothecina. This example illustrates that the quest for new Arcellinida diversity is far from being over and even the less expected ecosystems can bring new discoveries.