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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 speciesspecific 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.

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The minute intestinal trematode, , is an important parasite species that can infect humans and other mammals. This study investigated the outbreak of in thiarid snails in the lower part of the Chao Phraya Basin, Thailand by employing morphological and molecular-based methods. In development of a specific primer of , the PCR reaction was conducted with no cross-reaction to their hosts and other related trematode species. The highest level of sensitivity that could be amplified was 0.50 ng/μl and this was detected with only one egg in the sample. In terms of the epidemic results, the parapleurolophocercous cercaria infected only two species of thiarid snails ( and ) with an overall prevalence of 3.80% (23/605). The process of molecular identification revealed positive results indicating that eleven from twenty-three of parapleurolophocercous cercariae specimens in the lower part of the Chao Phraya Basin were. In conclusion, this study has developed a rapid detection method, which can discriminate from other parapleurolophocercous cercaria in intermediate snail hosts with a high level of sensitivity. Moreover, the high proportion of in parapleurolophocercous cercaria (47.83%) indicated that was the dominant species of this cercarial type and could infect cyprinoid fish in the lower part of the Chao Phraya Basin leading to public health problems in this area. Thus, a specific primer could be useful in the detection and surveillance of outbreaks in their hosts. Recognition of this has resulted in the creation of important prevention programs in these infected areas in the further study.