Fecal samples from 22 HIV-positive and 3 HIV-negative patients from Portugal with symptomatic diarrhea were diagnosed positive for microsporidia by microscopy, with most parasites detected significantly bigger than Enterocytozoon bieneusi and Encephalitozoon spp. Sequence characterization of the small subunit (SSU) rRNA gene identified a microsporidian parasite with 96% homology to two published Vittaforma corneae sequences. Phylogenetic analysis confirmed the genetic relatedness of this new microsporidian parasite to Vittaforma corneae as well as Cystosporogenes operophterae. Results of the study demonstrate the presence of a new human-pathogenic microsporidian species, which is responsible for significant number of infections in HIV-positive and HIV-negative patients in Portugal.