The work is described by microscopic analysis, the serological analysis (IFAT) and the molecular analysis of isolates from clinical samples (blood, faeces and urine) from ten domestic rabbits (Oryctolagus cuniculus), breed Maličký, four New Zealand domestic rabbits, 11 sows of breed Slo0076akian Improved White and 15 clinically healthy laboratory BALB/c mice. The aim of the study was to validate the suitability of species-unspecific primer pairs 530F and 580R for genotype determination of the Microsporidia strain and species-specific primer pairs ECUNF and ECUNR, SINTF and SINTR and EBIER1 and EBIEF1 for the determination of E ncephalitozoon cuniculi, Encephalitozoon intestinalis and Enterocytozoon bieneusi species for diagnostic purposes. Sequences of animals were compared with those from the GenBank database. In rabbits, two murine genotypes II and four canine genotypes III were identified. Genotype II was identified in mice. The Encephalitozoon intestinalis identified in the sample from swine showed no genetic heterogeneity.