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Abstract Background Cryptosporidium spp. are opportunistic intestinal protozoans with global distribution and are of great importance as zoonotic protozoans are common to humans and domestic animals, including cattle and calves. Identification and detection of parasite species using precise methods including molecular methods can be an effective step in treating and controlling parasites. Objectives This study aimed to investigate the prevalence of Cryptosporidium among breeding calves of Khorramabad city, Lorestan province, Western Iran, using PCR. Methods The faecal samples were taken from 181 healthy and diarrhoeal calves and after the Ziehl Neelsen Acid‐fast staining and microscopic evaluation, the genomic DNA was extracted for molecular evaluations. To detect Cryptosporidium species, specific primers targeting the SAM‐1 gene of Cryptosporidium and a commercial master mix were used for PCR. Results Out of 181 faecal samples of breeding calves in Khorramabad city, 9 samples (5%) were positive for Cryptosporidium spp. using the PCR method. Statistical analysis of the data showed that there was no significant statistical relationship between Cryptosporidium infection of the calves and variables of age, breed, type of water consumption, clinical signs of diarrhoea, and sampling location, while parasite infection had a significant relationship with calf gender so that all Cryptosporidium positive samples were from male calves (p ≤ 0.05). Conclusions Considering the presence of Cryptosporidium infection, the region's traditional grazing system, and the close relationship between livestock and humans, there is a possibility of human infection in the region. So preventive measures such as periodic animal testing with sensitive and accurate diagnostic techniques including PCR, pharmacological treatment of livestock, water hygiene and the use of industrial grazing instead of traditional grazing to improve the hygiene of food consumed by livestock are recommended.