To investigate the transmission of cryptosporidiosis in Portugal, Cryptosporidium hominis and Cryptosporidium parvum from HIV-infected patients, cattle, and wild ruminants were characterized by sequence analysis of the 60-kDa glycoprotein (GP60) gene. Fourteen subtypes within nine subtype families were identified, and three of the subtype families (If, IIb, and IId) were restricted or largely limited to Portugal. Parasites from cattle from various regions in Portugal and wild ruminants in Lisbon showed limited genetic heterogeneity (only two subtype families). All wild ruminants had the same subtype, which was also the predominant subtype in cattle all over Portugal and was found in nine HIV-infected patients in Lisbon. Two other C. parvum subtypes were only restricted to limited locations. In contrast, human parasites displayed 13 subtypes in nine subtype families, with most of the infections caused by parasites in Ib, IIa, IIc, and IId families. Two of the C. parvum subtype families (IIc and IIb) had only been found in humans. The high overall parasite diversity and high percentage of C. hominis infections attributable to Ib and C. parvum infections to IId represent unique characteristics of Cryptosporidium transmission in humans in Portugal.