BackgroundImportance of the amphizoic amoebaEntamoeba moshkovskiiis increasing in the study of amoebiasis as a common human pathogen in some settings. Limited studies are found on the genetic and phylogenetic characterization ofE. moshkovskiifrom India; hence remain largely unknown. In this study, we determined the prevalence and characterized theE. moshkovskiiisolates in eastern India.MethodsA three-year systemic surveillance study among a total of 6051 diarrhoeal patients from ID Hospital and BC Roy Hospital, Kolkata was conducted forE. moshkovskiidetection via a nested PCR system targeting 18S rRNA locus. The outer primer set detected the genusEntamoebaand the inner primer pair identified theE. moshkovskiispecies. The 18S rRNA locus of the positive samples was sequenced. Genetic and phylogenetic structures were determined using DnaSP.v5 and MEGA-X. GraphPad Prism (v.8.4.2), CA, USA was used to analyze the statistical data.Result4.84% (95%CI = 0.0433–0.0541) samples were positive forEntamoebaspp and 3.12% (95%CI = 0.027–0.036) were infected withE. moshkovskii. E. moshkovskiiinfection was significantly associated with age groups (X2= 26.01, P<0.0001) but not with gender (Fisher’s exact test = 0.2548, P<0.05). A unique seasonal pattern was found forE. moshkovskiiinfection. Additionally, 46.56% (95%CI = 0.396–0.537) were soleE.moshkovskiiinfections and significantly associated with diarrheal incidence (X2= 335.5,df = 9; P<0.0001). Sequencing revealed that the localE.moshkovskiistrains were 99.59%-100% identical to the prototype (GenBank: KP722605.1). The study found certain SNPs that showed a correlation with clinical features, but it is not necessarily indicative of direct control over pathogenicity. However, SNPs in the 18S rRNA gene could impact the biology of the amoeba and serve as a useful phylogenetic marker for identifying pathogenicE. moshkovskiiisolates. Neutrality tests of different coinfected subgroups indicated deviations from neutrality and implied population expansion after a bottleneck event or a selective sweep and/or purifying selection in co-infected subgroups. The majority of FSTvalues of different coinfected subgroups were <0.25, indicating low to moderate genetic differentiation within the subgroups of this geographical area.ConclusionThe findings reveal the epidemiological significance ofE. moshkovskiiinfection in Eastern India as the first report in this geographical area and expose this species as a possible emerging enteric pathogen in India. Our findings provide useful knowledge for further research and the development of future control strategies againstE. moshkovskii.