Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite’s DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.