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Background Mycoplasma gallisepticum (MG) infection is an economically important respiratory disease in the poultry production system worldwide. It is characterized by rapid transmission and causing many obstacles to poultry industries in different parts of Ethiopia. Methods A cross-sectional study was employed from January to September 2021 to estimate the seroprevalence and associated risk factors of MG in domestic layer chickens in large and small commercial poultry farms in Hawassa and Bishoftu area, Ethiopia. A total of 368 blood samples were collected. Data were analyzed using SPSS version-20, reported as percent prevalence, and Pearson's chi-square was used to assess the association between factors considered to have association with MG infection. The samples were processed by using an indirect ELISA (ProFLOKIBV, USA) test coated with antibody against MG. Results The total seroprevalence of MG infection was found to be 70.65%. Significant variation in environmental risk factors with seroprevalence was assessed and the higher prevalence of MG was significantly ( χ 2 = 14.42; p < 0.05) higher in layer chicken farms found in Bishoftu. Likewise, it was significantly observed in the adult chicken and commercial production system. There were significant difference between breeds and ages of birds with the occurrences of MG ( χ 2 = 19.60 and χ 2 = 17.46, respectively). Management related risk factors found around the types of farms were significantly different with the occurrences of MG (OR = 52.5; p < 0.05). Conclusion The evidence from seroprevalence of the MG infection in the current finding provides an indication of the eminence of infection in the study areas. Moreover, it provides an insight on the prevalence of MG infection and further molecular characterization of the organism needs to be conducted in the areas. Biosecurity measures combined with vaccination and sero-monitoring should also be implemented in the farms.

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