Aims: Wolbachia is an endosymbiont and a Gram-negative genus bacterium which has received the spotlight in the field of research studies due to its multiple capabilities to affect it hosts, including the bed bugs (Hemiptera: Cimcidae). While most investigations concentrated on the common bed bugs (Cimex lectularius), no published studies have yet to be done on molecular screenings of Wolbachia associated with tropical bed bugs (C. hemipterus). The present study was undertaken to screen Wolbachia infection from tropical bed bugs from Peninsular Malaysia. Methodology and results: We attempted to screen and characterize Wolbachia infections in tropical bed bugs from 22 different localities throughout Peninsular Malaysia using a molecular approach; multiple Polymerase Chain Reaction (PCR) assays with four sets of primer sequences. Conclusion, significance and impact of study Our findings yielded negative results of Wolbachia infections and, therefore, further confirmed that all bed bug samples from all localities in Peninsular Malaysia are free from Wolbachia infections. Our findings also suggested that the prevalence of Wolbachia in tropical bed bug populations in Peninsular Malaysia is very unlikely.