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Giardia duodenalis (G. duodenalis) is an intestinal flagellate protozoan, which infects in humans and animals. It can be causing giardiasis. The symptoms are acute or chronic diarrhea, abdominal pain, weight loss and dehydration. G. duodenalis can be transmitted by eating food or drinking water contaminated with infected stage cysts. Children who live and share facility in an orphanage are the high risk groups for infection. The objective of this paper is to study the prevalence and genotypes of G. duodenalis infection of asymptomatic individual in a child care center. This study was conducted in Mercy Centre orphanage, Klong Toey community. 289 stool samples were collected from children and childcare workers. Stool samples were extracted DNA. Small subunit ribosomal DNA (SSU-rDNA) and glutamate dehydrogenase (gdh) genes were amplified using Nested PCR. PCR products of gdh gene were cut using NlaIV and RsaI restriction enzymes. Genotypes were identified. The results of this study showed the prevalence of giardiasis in an orphanage, Klong Toey community, Thailand was 17.6% (51/289). PCR-RFLP analysis of 19 samples revealed that genotype AII was 63.2% (12/19), genotype BIV was 31.6% (6/19) and genotype BIII was 5.3% (1/19) respectively. Consequently, the study of prevalence and genotypes of G. duodenalis is beneficial for control planning of giardiasis in an orphanage, Klong Toey community.