Cryptosporidium is a protozoan parasite that causes gastrointestinal illness in a wide range of vertebrate hosts, including humans. Relatively little is known of the epidemiology of Cryptosporidium in fish. This study investigated the prevalence of Cryptosporidium in goldfish (Carassius auratus) (n=216) which were purchased from a retail aquarium and a fish farm in Perth, Western Australia. All samples were initially screened at the 18S locus by quantitative PCR (qPCR) and positives further analysed by nested PCR and sequencing at the 18S and actin loci. Further subtyping was conducted on human-infectious species at the glycoprotein 60 (gp60) locus. The overall prevalence by qPCR was 30.1% (65/216). Sequencing and phylogenetic analysis at the 18S locus (n=23) identified C. parvum (n=2), C. hominis (n=10) and a novel species (n=11), which was genetically distinct and most closely related to C. scopthalmi. (genetic distance = 10.1%). Sequencing at the actin locus (n=6) also confirmed the validity of the novel species (genetic distance =14.1%). Subtyping of three C. hominis isolates at the gp60 locus identified subtype 1bA10G2, which is the main C. hominis subtype involved in human outbreaks of cryptosporidiosis. Further research is required to characterise the novel species at the histological level and to determine the clinical impact of Cryptosporidium in goldfish.