Simple SummaryWe developed the DAS–ELISA to investigate the infection and prevalence of GyH1 in chickens and wild birds in China. We found that natural GyH1 infections were widespread in chickens and wild birds. Importantly, young chickens are more susceptible to GyH1, and local chicken species in China are genetically resistant to GyH1. In particular, we found a significantly higher GyH1–positive rate in wild birds than in chickens, implying that there may be a risk of GyH1 transmission from wild birds to chickens.AbstractGyrovirus homsa1 (GyH1) is an emerging pathogenic single–stranded circular DNA virus that leads to immunosuppression, aplastic anemia, and multisystem damage in chickens. However, the prevalence of GyH1 infection in chickens and wild birds remains unknown. Here, we developed a double–antibody sandwich enzyme–linked immunosorbent assay (DAS–ELISA) to investigate GyH1 infection in 8 chicken species and 25 wild bird species. A total of 2258 serum samples from chickens (n= 2192) in 15 provinces, and wild birds (n= 66) in Jinan Wildlife Hospital were collected from 2017 to 2021 in China. The GyH1–positive rates in chickens and wild birds were 9.3% (203/2192) and 22.7% (15/66), respectively. GyH1 was present in all flocks in 15 provinces. From 2017 to 2021, the positive rate ranged from 7.93% (18/227) to 10.67% (56/525), and the highest positive rate was present in 2019. Upon chicken age, the highest positive rate (25.5%) was present in young chickens (14–35 days old). Moreover, the GyH1–positive rate in broiler breeders (12.6%, 21/167) was significantly higher than that in layer chickens (8.9%, 14/157). This study shows that GyH1 has spread in chicken flocks and wild birds, and the higher GyH1–positive rate in wild birds indicates the risk of spillover from wild birds to chickens. Our study expanded the GyH1 epidemiological aspects and provided a theoretical basis for GyH1 prevention.