Two new microsporidia, Anostracospora rigaudi n. g., n. sp., and Enterocytospora artemiae n. g., n. sp. infecting the intestinal epithelium of Artemia parthenogenetica Bowen and Sterling, 1978 and Artemia franciscana Kellogg, 1906 in southern France are described. Molecular analyses revealed the two species belong to a clade of microsporidian parasites that preferentially infect the intestinal epithelium of insect and crustacean hosts. These parasites are morphologically distinguishable from other gut microsporidia infecting Artemia. All life cycle stages have isolated nuclei. Fixed spores measure 1·3×0·7 μm with 5–6 polar tube coils for A. rigaudi and 1·2×0·9 μm with 4 polar tube coils for E. artemiae. Transmission of both species is horizontal, most likely through the ingestion of spores released with the faeces of infected hosts. The minute size of these species, together with their intestinal localization, makes their detection and identification difficult. We developed two species-specific molecular markers allowing each type of infection to be detected within 3–6 days post-inoculation. Using these markers, we show that the prevalence of these microsporidia ranges from 20% to 75% in natural populations. Hence, this study illustrates the usefulness of molecular approaches to study prevalent, but cryptic, infections involving microsporidian parasites of gut tissues.