Despite economic importance of Tetranychidae, knowledge regarding diversity of spider mites in the Balkan Peninsula and Southeast Europe is incomplete, especially in protected natural areas. This study presents diversity of spider mites (Acari: Tetranychidae) collected over five growing seasons at 296 locations in 38 protected natural areas of Serbia. A total of 31 spider mite species were found, 10 from Bryobiinae and 21 from Tetranychinae. The species Eotetranychus fagi Zacher was recorded as new to Serbia and this record was also the first one for Southeast Europe. Spider mites were found on host plants in five basic types as well as many subtypes of terrestrial habitats, with woodland as the most dominant one. A total of 151 plant species from 44 families were recorded as hosts for spider mites including new world records: 60 new hosts for family Tetranychidae and 41 new hosts for 21 spider mite species. Host plants from Rosaceae family harbored the highest number of spider mite species (16). A considerable number of species was found on host plants from the families Betulaceae (11), Asteraceae (10) and Sapindaceae (10). Two cosmopolitan spider mites, Tetranychus urticae Koch and Tetranychus turkestani Ugarov & Nikolskii, were clearly distinguished with 67 (7 new) and 43 (13 new) recorded host species, respectively; among newly recorded hosts for Tetranychidae family, these two mite species were found on 27 and 12 hosts, respectively. After the two most common species, the most striking was the presence of Bryobia praetiosa Koch with 24 (4 new) recorded host plants, followed by Amphitetranychus viennensis (Zacher), Eotetranychus carpini (Oudemans) and Bryobia rubrioculus(Scheuten), with 21 (7 new), 20 (6 new) and 16 (2 new) hosts, respectively. The remaining tetranychids were found on 1–9 host plant species. This study provided the first insight into diversity of tetranychids in Serbian protected areas. Further research in this field should focus on mites from host plants representative of specific areas and habitats, including endangered, endemic and relict species.