The technique of pulsed field gel electrophoresis has been used to differentiate the chromosomes of Glugea stephani, Microgemma ovoidea, and two isolates of Spraguea lophii from different regions of Spain (Atlantic Ocean at Galicia and Mediterranean Sea at Catalonia). Although the karyotype of M. ovoidea could not be completely resolved, the range of sizes of its chromosomes was obtained (282-2601Kb). G. stephani showed 15 bands ranging 340-2654Kb and an estimated total size of the haploid genome of about 16775Kb. Both isolates of S. lophii had a very similar karyotype with 10 bands for the Atlantic isolate (range 266-1076 Kb) and 11 bands for the Mediterranean isolate (range 271-1120Kb). Digital processing of the gels allowed detection of non-homologous chromosome co-migration and revealed that both S.lophii isolates had 15 different kinds of DNA fragments, which could be interpreted by a homozygotic hypothesis (15 chromosomes) or a heterozygotic hypothesis (14 chromosome pairs, 13 pairs with the same size for both homologous, and a pair formed by a chromosome of about 423-490 Kb and its homologous of 353-495Kb).