BACKGROUND: Toxoplasmosis, having the significant consequences affecting mortality and quality of life, is still prevalent in various places throughout the world. The major gap in surveillance for Toxoplasma gondii infection among high-risk population, slaughterhouse workers, is an obstacle for the effective policies formulation to reduce the burden of toxoplasmosis in Myanmar. Therefore, this study aimed to assess the seroprevalence of toxoplasmosis and associated factors of seropositivity among slaughterhouse workers in Yangon Region, Myanmar. METHODS: A cross-sectional study that was conducted from June to November 2020 included 139 slaughterhouse workers involving at five main slaughterhouses under Yangon City Development Committee, Myanmar. The presence of IgG and IgM anti-T. gondii antibodies in serum was detected using the OnSite Toxo IgG/IgM Combo Rapid Test. A face-to-face interview was also performed using pretested structured questionnaires to obtain the detail histories: sociodemographic characteristics, level of knowledge, occupational factors, and environmental factors related to T. gondii infection. Bivariate logistic regression was used to determine the factors associated with T. gondii infection. RESULTS: Of all participants, the overall seroprevalence of anti-T. gondii was 43.9% (95% CI: 35.5-52.5%), of whom 98.4% (95% CI: 91.2-100.0%) were reactive only for IgG antibody and 1.6% (95% CI: 0.0-8.8%) were reactive for IgG and IgM antibodies. The significant factors associated with the seropositivity of T. gondii antibodies were blood transfusion history (OR: 5.74, 95% CI: 1.17-28.09), low level of knowledge (OR: 2.91, 95% CI: 1.46-5.83), contact with animal organs, muscles or blood (OR: 14.29, 95% CI: 1.83-111.51), and animals most frequently slaughtered (cattle) (OR: 3.22, 95% CI: 1.16-8.93). CONCLUSIONS: A high seroprevalence of toxoplasmosis was detected among slaughterhouse workers in Yangon Region and it raises a significant public health concern. Therefore, providing health education regarding toxoplasmosis, enforcement of personal hygiene practices in workplaces, the establishment of training for occupational hygiene, and commencement of the risk assessment and serological screening for toxoplasmosis are crucial to curtail the prevalence of T. gondii infection among slaughterhouse workers.