This paper summarises known data regarding floristic richness, distribution and habitat preferences of the charophyte algae (Charales) in the region of Vojvodina (Serbia). The historical records were collected from all available literature and herbarium data on charophytes in Vojvodina from the beginning of charophyte research in the 19th century to the present day. Moreover, detailed field investigations were carried out in the period from 2012 to 2014. The last study summarising knowledge about the distribution and ecology of charophytes (Charales) in Vojvodina was done in 1995, and nine species were listed therein. At the present time, 20 years later, 11 more species are known. Altogether, charophytes are represented in the flora of Vojvodina with 20 species, belonging to four genera: Chara, Nitellopsis, Nitella and Tolypella. All data were georeferenced and species distribution maps are given. Concerning habitat preferences, charophytes of Vojvodina are mainly found in three types of habitats, namely puddles and ruts in river floodplains, different kinds of excavation pits and periodically dredged canals. Most of the known localities are in the protected areas of Vojvodina.