Mycoplasma gallisepticum causes major health hazards in poultry birds in Bangladesh which results in huge economic losses every year. This study was carried out to estimate and analyze the prevalence of Mycoplasma gallisepticum (MG) infection in commercial layer chickens at Kishoreganj district of Bangladesh during the period from November 2018 to October 2019. A total of 505 serum samples from 94 commercial layer farms of Kishoreganj Sadar Upazila and Pakundia Upazila of Kishoreganj district were collected. Serum plate agglutination (SPA) was performed to detect the antibody against MG. Prevalence was found 73% in the Kishoreganj district by SPA test. MG was significantly (P<0.01) more prevalent in Pakundia Upazila 82% than Kishoreganj Sadar Upazila 61.11%. In case of season, winter season had significantly higher (X^2=30.94, p=0.000) prevalence of MG infection. In relation to age, seroprevalence of MG infection was highest 78% in birds of 65 weeks' age and lowest 71% in 6-25 weeks' age birds. Any significant (P>0.05) association was not found between flock size and seroprevalence of MG. Seroprevalence was highest in flock containing above 2600 birds. MG infection is prevalent in the chicken population of Kishoreganj district, Bangladesh. Measures should be taken for successful prevention and control of this disease in Bangladesh.