Epidemiological studies on species-specificEntamoebainfections are scarce due to the morphological similarity of pathogenicEntamoeba histolyticaand nonpathogenicE. disparandE. moshkovskii. The diagnosis ofE. histolyticais frequently based on coproantigen (E. histolytica-Gal/GalNAc lectin specific) detection by immunoassays. However, specificE. histolytica-lectin is not expressed in cysts, which are eliminated by asymptomatic individuals leading to false-negative results and an underestimation of amebiasis prevalence. Molecular techniques based on the amplification of parasite DNA have been shown to be a highly sensitive and specific method that allows the detection of differentEntamoebaspecies. This study aimed to assess the frequency of the species fromE. histolytica/dispar/moshkovskiicomplex by molecular and immunological techniques in individuals attended at a public health system in Salvador-Bahia, Brazil. A cross-sectional study involving 55,218 individuals was carried out. The diagnosis was based on microscopy revealingE. histolytica/dispar/moshkovskiicomplex. The species differentiation was performed byE. histolytica-specific antigen, serological evaluation and by molecular technique. The overall prevalence ofE. histolytica/dispar/moshkovskiicomplex determined by microscopy was approximately 0.49% (273/55,218).E. histolytica-specific antigen detection and molecular characterization returned 100% negativity forE. histolytica. However, serological evaluation returned an 8.9% positivity (8/90). In the stool samples analysed by PCR, it was not possible to identifyE. histolyticaandE. moshkovskii, although circulating IgG anti-E. histolyticahas been detected.