Cryptosporidium spp. infection is usually self-limited in immunocompetent hosts but can be severe and life threatening in children and in immunocompromised individuals including those with primary or acquired immunodeficiencies. One hundred and three faecal samples were collected from 35 hospitalised patients with different symptoms and tested for the presence of the parasite. Cryptosporidium oocysts were found in four of 35 patients (11.4%) using Ziehl-Neelsen staining of faecal smears and immunofluorescence assay, whereas 12 (34.3%) samples tested positive by nested polymerase chain reaction assay. Cryptosporidium DNA was detected in one bile sample but not in a liver tissue biopsy sample collected from a patient who suffered from sclerosing cholangitis. Sequence analysis of oocyst wall protein and beta-tubulin gene fragments revealed three different parasite species (Cryptosporidium hominis, Cryptosporidium meleagridis and Cryptosporidium parvum) in children with primary immunodeficiencies, whereas only C. parvum was found in immunocompetent individuals and in those with secondary immunodeficiencies. This study has revealed a high prevalence of Cryptosporidium infection in hospitalised patients in Poland and confirmed that molecular techniques enable a more sensitive detection of the parasite.