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Toxoplasmosis is a worldwide endemic zoonotic infection caused by the obligate coccidian parasite Toxoplasma gondii. To assess the seroprevalence of T. gondii infection among hospital population of Hospital Universiti Sains Malaysia (HUSM), a cross-sectional study was conducted using serum samples of 102 participants. Samples were screened for the presence of anti-Toxoplasma IgG and IgM antibodies by enzyme-linked immunosorbent assay (ELISA). Only one sample recorded as IgM positive (0.98%) (1/102; 95% CI = 0.02-5.34%), whereas 44.12% (45/102; 95% CI = 34.29-54.29%) were IgG positive. The study showed that seroprevalence of toxoplasmosis significantly differs between age groups (p < 0.001), with high prevalence of T. gondii infection (37.8%) among patients aged less than one month. Moreover, the study indicated a significant difference in the prevalence of toxoplasmosis between males and females. Additionally, a significant dependent relationships were observed (p = 0.003), when the reasons of toxoplasma serology request was considered, and the highest prevalence (59.6%) observed in congenital toxoplasmosis. In conclusion, the seroprevalence of toxoplasmosis in our study group was high. The study provides preliminary information about the seroprevalence and epidemiology of T. gondii infection among the hospital population in HUSM.