Objective: To explore the genetic variation and differentiation of 2 echinostomes from genus Echinostoma, i.e. Echinostoma revolutum (E. revolutum) and Echinostoma malayanum (E. malayanum) from Khon Kaen Province, Thailand. Methods: These parasites were compared at 22 enzymes encoding a presumptive 30 loci by using multilocus enzyme electrophoresis (MEE) technique. Results: Twenty-two loci can be used as diagnostic markers to differentiate these 2 species. E. revolutum and E. malayanum had fixed genetic differences at 70% of loci, whereas both species had fixed genetic differences from the liver fluke, Opisthorchis viverrini at 91% of loci. Intraspecific variation within a population of E. revolutum was observed at 5 polymorphic loci. Conclusions: MEE is a powerful technique to investigate genetic variation and differentiation of E. revolutum and E. malayanum.