We applied a two-dimensional pulsed-field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi, termed D (strain III) and F (strain II). Data obtained for BssHII and Mlul restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain I, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitolic recombination between conserved regions.