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Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Gallid herpesvirus 1 (GaHV-1), a.k.a infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. Despite the clinical signs on the field suggestive of ILT, it has long been considered nonexistent and a disease of no concern in Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021 in three selected zones of the Amhara region (Central Gondar, South Gondar, and West Gojjam zones), Ethiopia, with the objective of estimating the seroprevalence of ILTV in chickens and identifying and quantifying associated risk factors. A total of 768 serum samples were collected using multistage cluster sampling and assayed for anti-ILTV antibodies using indirect ELISA. A questionnaire survey was used to identify the potential risk factors. Of the 768 samples, 454 (59.1%, 95% CI: 0.56-0.63) tested positive for anti-ILTV antibodies. Mixed-effect logistic regression analysis of potential risk factors showed that local breeds of chicken were less likely to be seropositive than exotic breeds (OR: 0.38, 95% CI: 0.24-0.61). In addition, factors such as using local feed source (OR: 6.53, 95% CI: 1.77-24.04), rearing chickens extensively (OR: 1.97, 95% CI: 0.78-5.02), mixing of different batches of chicken (OR: 14.51, 95% CI: 3.35-62.77), careless disposal of litter (OR: 1.62, 95% CI: 0.49-4.37), lack of house disinfection (OR: 11.05, 95% CI: 4.09-47.95), lack of farm protective footwear and clothing (OR: 20.85, 95% CI: 5.40-80.45), and careless disposal of dead chicken bodies had all been associated with increased seropositivity to ILTV. Therefore, implementation of biosecurity measures is highly recommended to control and prevent the spread of ILTV. Furthermore, molecular confirmation and characterization of the virus from ILT suggestive cases should be considered to justify the use of ILT vaccines., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Mastewal Birhan et al.)