Background:Different genotypes ofEchinococcus granulosus s.l.infect humans and ungulate animals, causing cystic echinococcosis. Simultaneous isoenzyme, as well as molecular characterizations of this parasite, has not yet been investigated in Iran. The present study aimed to evaluate the isoenzyme pattern of theE. granulosus s.s.andE. canadensisgenotypes in Iran.Methods:A total of 32 (8 humans and 24 animals) cystic echinococcosis cysts were isolated from Shiraz, Tehran, Ilam, and Birjand from May 2018 to December 2020. The DNAs were extracted and their genotypes were determined by molecular methods. Enzymes were extracted from the cysts and subjected to polyacrylamide gel electrophoresis. The activities of G6PD, MDH, ME, NH1, and ICD were examined in the cyst samples using isoenzyme method and compared it with the genotyping findings.Results:DNA sequence analysis of the samples showed that the specimens contained 75%E. granulosus s.s.(G1) and 25%E. canadensis(G6) genotypes. The isoenzyme pattern of ICD in both genotypes produced a six-band pattern with different relative factors. The G6PD also produced two bands with different relative migrations in both genotypes. The MDH and NH1 systems revealed a two-band pattern, while only one band was generated in the ME enzyme in theE. granulosus s.s.genotype. In theE. canadensis, the MDH and NH1 enzymes showed one band, and the ME enzyme represented a two-band pattern.Conclusion:Our findings suggest thatE. granulosus s.s.andE. canadensisgenotypes have entirely different isoenzyme patterns for NH1, G6PD, MDH, and ME.