Echinostoma revolutum is known as a significant intestinal trematode in various species of animals and humans. It presents complexities in terms of both the morphological and molecular biological data. This is the first study of the application of Cytochrome B gene (CYTB) as a target for studying the phylogeny and designing species-specific primer of E. revolutum. Adult trematodes were harvested from experimentally infected hamsters at 18 days of post-infection. Each worm was identified based on their morphological appearance. The novel CYTB primers were designed from other Echinostoma species to initially amplify CYTB region in E. revolutum. All sequence data of E. revolutum in five provinces of Central Thailand were used as the target for designing the species-specific primer for E. revolutum. The results revealed that CYTB gene can separate E. revolutum into two sister groups by geographical distribution, comprising the eastern and western area groups. Moreover, it also separates E. revolutum from other Echinostoma species, including two sibling species; E. caproni and E. paraensei. In addition, we developed the high performance species-specific primer of E. revolutum. It can detect DNA from a single egg, as well as cercaria, metacercaria and adult stages of this trematode with no cross-reactions to other trematodes and their hosts. Therefore, this research is a positive initial step for the future study of E. revolutum CYTB. The future studies based on this gene should be continued with all species in revolutum complex to overcome the problems of systemic classification that arise in this complex group.