Entamoeba species are commonly detected in stool samples of Orang Asli due to their substandard living conditions and poor hygiene. Among the Entamoeba spp., Entamoeba histolytica is the only known primary pathogenic species. This study determined the prevalence and distribution of anti-amoebic IgG antibody among Orang Asli in Peninsular Malaysia. The results would reflect the prevalence of amoebiasis in the population. This study analysed a total of 375 serum samples from archives of two Orang Asli projects conducted between 2011 and 2014. They were from six different states in Malaysia, namely Johor, Kedah, Kelantan, Pahang, Perak, and Selangor. Anti-amoebic IgG antibody was detected using an enzymelinked immunosorbent assay (ELISA) with crude soluble antigen produced from axenically grown E. histolytica trophozoites. From the analysis, the overall seropositivity was approximately 71% (266/375), while the seropositivity rates for each of the three Orang Asli tribes i.e. Senoi, Negrito and Proto-Malay, were 66% (137/208), 92% (103/112), and 43% (17/ 41) respectively. Orang Asli from Kedah [95% (52/55)] showed the highest seropositivity, followed by Kelantan [79% (54/68)], Perak [73% (78/107)], Pahang [60% (57/95)], Selangor [56% (14/25)], and Johor [48% (10/21)]. Orang Asli from rural [76% (192/254)] and peripheral urban [65% (69/106)] areas showed significantly higher seropositivity (p=0.002) than those from urban areas [36% (4/11)]. The high prevalences of anti-amoebic IgG antibody in these Orang Asli populations comprised both active and past infections. This study provides current insights of amoebiasis in selected Orang Asli settlements in Peninsular Malaysia. The high seropositivity of anti-amoebic IgG antibody suggests that the settlements are endemic for amoebiasis and there is a high risk of acquiring E. histolytica infection among the dwellers.