Giardia duodenalis is one of the most prevalent human intestinal parasite, with children living in developing countries being particularly at risk of infection. The occurrence and molecular diversity of G. duodenalis was investigated in stools specimens from 307 individuals aged one to nineteen years in Colombia. Samples were collected in three educational establishments (n: 163) and two hospital laboratories (n: 144) from urban and rural areas. Feces were concentrated using a biphasic sedimentation method and wet mounts of the sediment were examined by light microscopy. G. duodenalis assemblages and sub-assemblages were determined on positive samples by PCR of the triose phosphate isomerase (tpi), β-giardin (bg) and small-subunit (ssu) rRNA genes. G. duodenalis infection was detected by microscopy in 23 individuals (7.5%). The protozoan was more prevalent among specimens collected in educational establishments (11.6%) than in those obtained from hospital laboratories (2.8%). Infection was most common in individuals from urban areas and children aged 1–5 years. No significant association between diarrhea and infection could be demonstrated. Twenty Giardia-positive samples were successfully allocated to assemblage B (n: 11), sub-assemblage AII (n: 7), and assemblage A (n: 2). Results indicate the potential for transmission of G. duodenalis infection in children attending educational establishments and individuals from urban areas, where transmission seems to be primarily anthroponotic.