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WE&H group: Carles Conejero, Carlos González-Crespo, Cristina Garrido, Diana Gassó, Diana Andrea Murillo, Emmanuel Serrano, Gregorio Mentaberre, Irene Torres-Blas, Josep Estruch, Josep Pastor, Jorge Ramón López-Olvera, María Escobar-González, Marta Valldeperes, Montse Mesalles, Omar López, Raquel Álvarez, Rafaela Cuenca, Roser Velarde, Santiago Lavín., Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals., This work was supported by Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, under project PI19CIII/00 029. Sample collection in Catalonia was supported by the Council of Barcelona through contracts 13/051, 15/0174, 16/0243 and 16/0243-00-PR/01, from the Spanish Ministry of Economy and Competitiveness under research grants CGL2012- 40043-C02-01, CGL2012-40043-C02-02, and from the Spanish Ministry of Science and Innovation under research grant CGL2016-80543-P PID2020-115046GB-I00, and USDA-ARS Project No: 8042 -32000-112 -00-D., Antonio Rivero-Juárez is the recipient of a Miguel Servet Research Contract by the Ministerio de Ciencia, Promoción y Universidades of Spain (CP18/00111). D.G.B. is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. A.D. is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities., Medical Mycology