Cryptosporidia are important protozoan parasites of vertebrates, and a number of species and genotypes, with different host ranges, have been described. In this study a protocol was established for the detection and the genetic characterization of Cryptosporidium spp. isolated from various types of surface waters (rivers, creeks, lakes, sewage plant in- and outlets and swimming pools) from the area between Zurich (Switzerland) and Munich (Germany). Cryptosporidium oocysts were isolated by continuous-flow-centrifugation and immunomagnetic separation (IMS). A novel nested PCR combined with direct sequencing of the amplicon which spans a variable region of the 18S rRNA allowed characterization of species and genotypes. Cryptosporidium spp. were detected in 23 of the 68 water samples investigated. Almost half of these isolates represent species and genotypes known to be pathogenic to man, namely C. parvum ‘bovine genotype’ (from 6 samples) and C. parvum ‘human genotype’ (4). Furthermore, we identified C. muris ‘genotype A’ (3), C. muris ‘genotype B’ (6), C. baileyi (1) as well as 3 novel Cryptosporidium genotypes. Our results confirm the ubiquitous presence of Cryptosporidium oocysts in surface waters. The detection of a variety of species and genotypes stresses the importance that molecular characterization is indispensable before drawing conclusions of medical or epidemiological significance.