Simple SummaryToxoplasma gondiiis an emerging foodborne parasite disease causing morbidity and mortality worldwide. The coccidian parasite,Toxoplasma gondii, has an exceptionally broad spectrum of intermediate hosts. Felids are the only definitive host responsible in producing oocysts that are highly resistant to environmental elements. Toxoplasmosis is common leading to public health concerns and economic losses to the animal industry. In the current study, serum and tissue samples from village chickens and pigs were examined. In chickens, 7.6% of serum and 14.0% of tissue samples were detected withT. gondii.On the other hand, 3.0% of serum and 5.8% of pig tissue samples were positive withT. gondii.Six unique DNA sequences were isolated from the tissue samples. The risk factor findings, which are the first study in Malaysia, emphasised the necessity for covering feed storage and tightening the biosecurity in farm to lower the exposure of village chickens and pigs to the parasite.AbstractToxoplasma gondiiis an important zoonotic foodborne parasite capable of infecting almost all warm-blooded animal species worldwide. Toxoplasmosis is usually acquired via ingestion of undercooked infected animal tissues resulting in life-threatening consequences for unborn foetus and immunocompromised individuals. A cross-sectional study was carried out to determine the prevalence ofT. gondiiinfection, its associated risk factors in farms, and haplotypes isolated from the native village chicken and pig populations in Peninsular Malaysia. The seroprevalence ofT. gondiiin village chickens at the animal level was low at 7.6% (95% CI: 4.60–11.60), while at the farm level, it was 52.0% (95% CI: 31.30–72.20). For pigs, the animal-level seroprevalence ofT. gondiiwas 3.0% (95% CI: 1.60–5.10), while the farm-level, it was 31.6% (95% CI: 12.60–56.60). The PCR-based DNA detection on meat samples from chickens (n= 250) and pork (n= 121) detected 14.0% (95% CI: 9.95–18.9) and 5.8% (95% CI: 2.4–11.6) positive, respectively. Six uniqueT. gondiihaplotypes were isolated from the tissue samples. Multivariable logistic regression analysis showed that feeding the chickens farm-produced feeds and allowing wild animals access to pig farms were significant determinants for farm-level seropositivity. Providing hygienic and good quality feeds to chickens and increasing biosecurity in pig farms through prevention of access by wildlife may reduce the risk of transmission ofT. gondiiinfection in the local chickens and pig farms.