Gastric habronemosis of horses caused by Habronema microstoma and Habronema muscae (Nematoda, Spirurida) is characterized by catarrhal gastritis, diarrhoea, progressive weight loss and ulcers. Despite its importance in the equine industry and in clinical practice, knowledge of the epidemiology of this infection is still incomplete as diagnosis in live animals is challenging. A two-step semi-nested PCR assay using ribosomal DNA (rDNA) markers has recently been used for the molecular diagnosis in vivo of gastric habronemosis based on the detection of H. microstoma and/or H. muscae DNA in equine faeces. To evaluate the field efficacy of this assay, a molecular epidemiological survey was carried out on equid gastric habronemosis in central Italy. One hundred and fifty-three individual faecal samples were collected from live native horses and subjected to both coprological examination and the two-step semi-nested PCR. When flotation procedures were performed no horse tested positive for Habronema spp. larvated eggs while 96 animals (61.2%) were positive for other endoparasites (i.e. strongyles, oxyurids, ascarids). Two-step semi-nested PCR detected 86 samples (53.6%) that were positive for H. microstoma and/or H. muscae DNA. H. microstoma prevalences showed statistically significant differences; the highest prevalence was observed in horses infected by other gastrointestinal parasites and concomitantly by H. muscae. No statistical differences were found between the prevalence of Habronema spp. infection and sex, age, breeding management, and antiparasitic treatments. This field survey provided further information on habronemosis and its epidemiology.