Abstract Background The prevalence of anti-platelet factor 4 (PF4)/polyanionic antibodies occurring after vaccination with ChAdOx1 nCoV-19 is low. Most of these antibodies are not associated with vaccine-induced thrombotic thrombocytopenia. It remains unknown whether these antibodies are preexisting or occur as a result of vaccination. In this study, we demonstrated the incidence of anti-PF4/polyanionic antibodies, thrombocytopenia, and thrombosis after vaccination with ChAdOx1 nCoV-19 in a large cohort of Thais. Methods We conducted a prospective study in a cohort of health care workers and members of the general population who received COVID-19 vaccination with ChAdOx1 nCoV-19. Blood collection for complete blood count, D-dimer, and anti-PF4/polyanionic antibodies was performed before vaccination (day 0), day 10, and day 28 after vaccination. Anti-PF4/polyanionic antibodies were detected using enzyme-link immunosorbent assay (ELISA). Functional assay was performed for all positive ELISA tests. Results A total of 720 participants were included in the study. 214 participants received both the first and second doses, 91 participants received only the first, 51 received only the second, and 364 received the third booster dose of ChAdOx1 nCoV-19. Median age was 42 years (IQR, 34–53). 67% of participants were female. Three participants developed seroconversion, yielding an incidence of vaccination-induced anti-PF4/polyanionic antibodies of 0.42% (95% confidence interval 0.08, 1.23). Fourteen (1.9%) participants had preexisting anti-PF4/polyanionic antibodies before the vaccination but their optical density of anti-PF4/polyanionic antibodies did not significantly increase over time. None of the anti-PF4/polyanionic positive sera induced platelet aggregation. Abnormal D-dimer levels following vaccination were not different among the positive and negative anti-PF4/polyanionic groups (11.8% vs. 13.2%, p = 0.86). Thrombocytopenia occurred in one person with negative anti-PF4/polyanionic antibodies. No clinical thrombosis or bleeding occurred. Conclusion We found a low incidence of seroconversion of anti-PF4/polyanionic antibodies after vaccination with ChAdOx1 nCoV-19 in Thais. Most of the anti-PF4/polyanionic antibodies were preexisting and did not significantly increase after vaccination with ChAdOx1 nCoV-19. Following vaccination, some participants with anti-PF4/polyanionic antibodies had elevated D-dimer levels, while only one developed thrombocytopenia and no thrombotic events were observed.