Dear Editor-in-Chief, In Annals of Parasitology 2021, 67(1), 55-65, a paper entitled "Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt" was published with great interest [1]. After reading the article carefully and critically, we think some points should be noted. Fasciola species are meiotically functional diploid, can produce sperm and temporarily and store in the seminal vesicles. This type is named spermic fluke [2]. On the other hand, intermediate Fasciola with morphological characteristics intermediates between F. hepatica and F. gigantica with no sperm or aspermic and no sperm in seminal vesicles. However, this is also seen in older flukes [3-5]. It seems that morphological studies based on spermatogenesis ability were necessary for this study. Also, this parasite's anthelmintic resistance is due to aspects of biology, and population structure depends on genetic diversity [6]. We question whether there are any documents about and sequences of mitochondrial markers as COX (Cytochrome Oxidase) and NAD (Nicotinamide Adenine Dinucleotide) to analyze intraspecific phylogenetic relationship in addition to nuclear gene? In Table 3, the pairwise distances between three groups of Fasciola spp. from different livestock animals were low, ranging from 0.004 to 0.01 with an overall mean of 0.008. Genetic diversity is described as a tendency of genetic characteristics to vary and serves as a way for the population to adapt to changing hosts and environments [7]. The nature of the nuclear gene (ITS) is instability. It is better to use mitochondrial sequence data to compare diversity. Also, genetic discrimination grade from infra population to meta population is annotated by Fst value ranging; 0 to 1. Fst values between 0-0.05 indicated a low genetic differentiation population [8]. It seems that by calculating Fst and showing the gene migration based on mitochondrial sequences data of specimens, this study's species population will be obtained. Also, Tajima's D and Fu's F in all loci populations based on GenBank data may show the Fasciola haplotypes' population proximity. Here we recommend, that Omar et al. [1] studies that molecular phylogeny with mitochondrial DNA efectively used for appropriate diferentiation of haplotypes and spermatogenic ability by carmen allium staining helps them find the physiological aspects. Of course, more prominent populations are needed to find intermediate types. [1] Omar M.A, Elmajdoub L.O., Ali A.O., Ibrahim D.A., Sorour S.S., Al-Wabel M.A., Suresh M., Metwally A.M. 2021. Genetic characterization and phylogenetic analysis of Fasciola species based on ITS2 gene sequence, with first molecular evidence of intermediate Fasciola from water buffaloes in Aswan, Egypt. Annals of Parasitology 67: 55-65. doi:10.17420/ap6701.312 [2] Sanderson A. 1953. Maturation and probable gynogenesis in the liver fluke, Fasciola hepatica L. Nature 172: 110-112. doi:10.1038/172110a0 [3] Hayashi K., Ichikawa-Seki M., Mohanta U.K., Singh T.S., Shoriki T., Sugiyama H., Itagaki T. 2015. Molecular phylogenetic analysis of Fasciola flukes from eastern India. Parasitology International 64: 334-338. https://doi.org/10.1016/j.parint.2015.04.004 [4] Ichikawa-Seki M., Tokashiki M., Opara M.N., Iroh G., Hayashi K., Kumar U.M., Itagaki T. 2017. Molecular characterization and phylogenetic analysis of Fasciola gigantica from Nigeria. Parasitology International 66: 893-897. doi:10.1016/j.parint.2016.10.010 [5] Rouhani S., Raeghi S., Mirahmadi H., Fasihi Harandi M., Haghighi A., Spotin A. 2017. Identification of Fasciola spp. in the east of Iran, based on the spermatogenesis and nuclear ribosomal DNA (ITS1) and mitochondrial (ND1) genes. Archives of Clinical Infectious Diseases 12:e57283. doi:10.5812/archcid.57283 [6] Hodgkinson J., Cwiklinski K., Beesley N., Paterson S., Williams D., Devaney E. 2013. Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica. Parasitology 140: 1523. doi:10.1017/S0031182013000528 [7] Bozorgomid A., Rouhani S., Harandi M.F., Ichikawa- Seki M., Raeghi S. 2020. Genetic diversity and distribution of Fasciola hepatica haplotypes in Iran: molecular and phylogenetic studies. Veterinary Parasitology: Regional Studies and Reports 19: 00359. [8] Rouhani S., Raeghi S., Spotin A. 2017. Spermatogenic and phylo-molecular characterizations of isolated Fasciola spp. from cattle, North West Iran. Pakistan Journal of Biological Sciences 20: 204-209.