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From September 2020 to April 2021 a mosquito monitoring activity was carried out in the Caffarella Valley within the Appia Antica Regional Park in Rome, a very popular urban park completely surrounded by densely populated neighborhoods. During this entomological survey, we collected 798 mosquito larvae that were identified by morphology and by molecular tools. Out of these samples, eight taxa have been identified morphologically or molecularly in case of complex of species, belonging to five genera: Culex pipiens (49.1%, n=392), Culiseta annulata (16%, n=128), Culiseta longiareolata (13.1%, n=105), Anopheles claviger (11.5%, n=92), Aedes albopictus (5.5%, n=44), Anopheles maculipennis s.s. (2%, n=16), Culex theileri (0.7%, n=6), Uranotaenia unguiculata (1.8%, n=15). The noteworthy datum of this survey is the finding of Cx. theileri, a very common species in ponds and marshes in natural and rural environments, but never recorded before in Rome, and also the record of Ur. unguiculata, previously reported in Rome but never observed in this park. Caffarella Valley confirms its high degree of naturalness and high biodiversity in terms of mosquito species together to its particular location as a very popular urban park, completely surrounded by densely populated neighborhoods. [ABSTRACT FROM AUTHOR]