The genomes of microsporidia in the genus Encephalitozoon have been extensively studied for their minimalistic features, but they have seldom been used to investigate basic characteristics of the biology of these organisms, such as their ploidy or their mode of reproduction. In the present study, we aimed to tackle this issue by mapping Illumina sequence reads against the genomes of four strains of E. cuniculi. This approach, combined with more conventional molecular biology techniques, resulted in the identification of heterozygosity in all strains investigated, a typical signature of a diploid nuclear state. In sharp contrast with similar studies recently performed on a distant microsporidian lineage (Nematocida spp.), the level of heterozygosity that we identified across the E. cuniculi genomes was found to be extremely low. This reductive intraindividual genetic variation could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce.