Globally, Giardia duodenalis is probably the most common intestinal protozoan parasite infecting humans and it appears also to be common in some zoo-housed primates. Infected zoo animals present a risk for potential spill-over of zoonotic pathogens to co-residing animals, staff and visitors. Using quantitative PCR, this study compared Giardia spp. prevalence and infection intensity in wild and zoo-housed ring-tailed lemurs Lemur catta. Infection intensity of zoo-housed ring-tailed lemurs (prevalence=88.6%, median Ct value=31.1, IQR=27.1-34.5) was significantly higher (P>0.01) than in wild ring-tailed lemurs (prevalence=20.0%, median Ct value=37.7, IQR=37.5-38.7), where little or no Giardia was found. Comparison of the enclosure designs showed both a higher prevalence and significantly higher intensity (P>0.005) of Giardia infections in zoos with walk-through enclosures (prevalence=89%, median Ct value=28.6, IQR=26.5-32.3) compared to traditional enclosures (prevalence=65%, median Ct value=35.2, IQR=33.3-37.8), but there was substantial variation within groups. The potentially zoonotic G. duodenalis assemblage B was identified in samples from five zoos. These findings suggest that ring-tailed lemurs may be asymptomatic carriers of G. duodenalis and a higher parasitic load might occur in lemurs held in walk-through enclosures.