Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes.