Giardia duodenalis, a flagellated protozoan, represents a common cause of gastroenteritis in Ethiopia, however very little information is available on the epidemiology and transmission routes of this pathogen, and a genetic characterization of the parasite has never been attempted in this country. The aim of this study was the genetic analysis of human isolates of G. duodenalis collected in different localities across the country, both from urban and rural areas. A fragment of the beta-giardin gene was amplified by nested PCR and analyzed by restriction and sequence analyses. Of the 59 isolates examined, 31 (52%) were typed as assemblage A and 13 (22%) as assemblage B. A strong correlation between the presence of symptoms and infection with assemblage B was observed. The remaining 15 (25%) isolates were typed as mixed infections by PCR-RFLP, specifically, A+F (in seven isolates) and A+B (in eight isolates). Sequencing of the A+F products confirmed the presence of assemblage F in three isolates, whereas the remaining four were identified as assemblage A. The detection of assemblage F, a cat-specific assemblage that to date has not been associated with human infections, was not able to be confirmed by the analysis of two commonly used markers (small subunit ribosomal RNA and triosephosphate isomerase). The analysis of the one isolate that was successfully amplified with the glutamate dehydrogenase primers unambiguously identified it as G. duodenalis, yet it was distinct from the established A and F sequences; thus the exact genetic identity of these isolates remains unclear.