West Nile virus (WNV) is a zoonotic and arthropod-borne flavivirus that can be transmitted by biting infected mosquitoes. The horse is a dead-end host that is highly susceptible to the disease, which may develop nervous signs. A previous orang asli and companion birds report revealed seropositivity against WNV in Malaysia. However, thus far, there is no data reported on the status of WNV infection in a horse in Malaysia which makes this study serve as preliminary research. Although WNV is not endemic in Malaysia yet, most breeds imported to Malaysia were derived from WNV endemic countries. By considering these facts, this study aims to detect the presence of WNV in horses via serological and molecular methods by using competitive ELISA (ID Screen ® West Nile Competitive Multi-species) and rt-PCR, respectively. Convenient sampling was performed by obtaining serum and oropharyngeal swabs from 20 horses in 3 different states: Selangor, Putrajaya, and Kuala Lumpur. By using ELISA, 19/20 samples were positive against WNV anti-prE antibodies. One step rt-PCR targeting the highly conserved gene between capsid and pre-membrane revealed that seven samples were positive (Gender: Mare=2, Gelding=5; Location: Putrajaya=3, Kuala Lumpur=4, Selangor=0 and Breed: Thoroughbred=3, Warmblood=4). The positive bands were submitted to First Base Laboratory for sequencing analysis and revealed 98-99% homologous to WNV strain from South Africa, the USA, Hungary, Italy and North Europe. The phylogenetic tree revealed that the positive samples aligned with strains found in USA and Austria, Central Europe. In conclusion, this study confirmed that WNV was successfully being detected and this proved that there was exposure to WNV among equine populations in the central part of Malaysia.