Giardia duodenalis is one of the most important human enteric parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. However, there is limited information on the prevalent genetic variability of G. duodenalis in Syria. This study aimed to evaluate the predominance of G. duodenalis assemblages/sub-assemblages causing humans infection in the city of Damascus and its suburbs. 40 symptomatic giardiasis patients were recruited in this study. Fecal samples were genotyped using PCR/RFLP assay targeting the β-giardin and glutamate dehydrogenase (gdh) genes. HaeIII, BspL1 and RsaI restriction enzymes were used to differentiate between G. duodenalis assemblages/sub-assemblages. Our data showed that 65% of isolates were of assemblage A; 45% belonged to sub-assemblage AII and 20% to sub-assemblage AI. Assemblage B was detected in 27.5% of isolates; 12.5% fit in sub-assemblage BIV, 5% fit in sub-assemblage BIII and 10.5% fit in Discordant genotype BIII/BIV. Mixed genotypes (AII+BIII and AI+BIV) were identified in 3 isolates (7.5%). Significant correlation was found between Giardia AII sub-assemblage and weight loss symptom (P-value=0.05) as well as between contact with domestic animals (cats, P-value=0.027). Moreover, a significant correlation was found between sub-assemblage AI and livestock breeding (P-value=0.000). In conclusion genotyping of human Giardia duodenalis isolates suggests anthroponotic transmission for the route of infection in Damascus and its suburbs. Further studies are needed to screen a wide geographic areas in Syria and to estimate the prevalence of G. duodenalis infection in our population.