BackgroundIntestinal parasitic infections are considered a serious public health problem and widely distributed worldwide, mainly in urban and rural environments of tropical and subtropical countries. Globally, soil-transmitted helminths and protozoa are the most common intestinal parasites. Blastocystis sp. is a highly prevalent suspected pathogenic protozoan, and considered an unusual protist due to its significant genetic diversity and host plasticity.Methodology/main findingsA total of 294 stool samples were collected from inhabitants of three rural valleys in Rio de Janeiro, Brazil. The stool samples were evaluated by parasitological methods, fecal culture, nested PCR and PCR/Sequencing. Overall prevalence by parasitological analyses was 64.3% (189 out of 294 cases). Blastocystis sp. (55.8%) was the most prevalent, followed by Endolimax nana (18.7%), Entamoeba histolytica complex (7.1%), hookworm infection (7.1%), Entomoeba coli (5.8%), Giardia intestinalis (4.1%), Iodamoeba butchilii (1.0%), Trichuris trichiura (1.0%), Pentatrichomonas hominis (0.7%), Enterobius vermicularis (0.7%), Ascaris lumbricoides (0.7%) and Strongyloides stercoralis (0.7%). Prevalence of IPIs was significantly different by gender. Phylogenetic analysis of Blastocystis sp. and BLAST search revealed five different subtypes: ST3 (34.0%), ST1 (27.0%), ST2 (27.0%), ST4 (3.5%), ST8 (7.0%) and a non-identified subtype.Conclusions/significanceOur findings demonstrate that intestinal parasite infection rates in rural areas of the Sumidouro municipality of Rio de Janeiro, Brazil are still high and remain a challenge to public health. Moreover, our data reveals significant genetic heterogeneity of Blastocystis sp. subtypes and a possible novel subtype, whose confirmation will require additional data. Our study contributes to the understanding of potential routes of transmission, epidemiology, and genetic diversity of Blastocystis sp. in rural areas both at a regional and global scale.