Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250-300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3-16 years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia.