BackgroundProtozoan pathogens from the genusCryptosporidiumcause the diarrhoeal disease cryptosporidiosis in humans and animals globally. Freshwater biota could act as potential reservoirs or zoonotic sources ofCryptosporidiuminfections for livestock and people, butCryptosporidiumoccurrence in aquatic biota is largely unexplored. The aim of this study was to investigate the occurrence ofCryptosporidiumin a range of freshwater organisms in upland rivers across England and Wales.MethodsFish were sampled by electrofishing, invertebrate larvae by kick sampling and the otterLutra lutraand minkMustela visonthrough faecal samples collected opportunistically as part of a nation-wide study. PCR targeting the small subunit ribosomal RNA gene was used to detectCryptosporidiumspecies.ResultsCryptosporidiumoccurred in just 0.8% of all the samples and in none of 73 samples from nine invertebrate genera.Cryptosporidiumwas detected in two of 2/74 fish samples (2.7%), both salmonids, and in 2/92 otter faecal samples (2.17%), but there were no positive samples in mink (0/24) or the bullheadCottus gobio(0/16).ConclusionsLow detection rate of human-infectiveCryptosporidiumspecies in aquatic fauna indicates they may present a low risk of contamination of some upland freshwaters.Graphical Abstract