Abstract Background Parasitic infections pose significant threats to humans’ and animals’ well-being worldwide. Among these parasites, Halicephalobus spp., a genus of nematodes, has gained attention due to its ability to cause severe infections in various animal species, including horses. Objective This study aimed to determine the prevalence of Halicephalobus spp., specifically focusing on Halicephalobus gingivalis in horses. Materials and methods In July 2022, a cross-sectional study was conducted in northern Iran to determine the prevalence of Halicephalobus spp. Using standard coprological techniques, 141 fecal samples from randomly selected horses were analyzed for GI helminth eggs. The Halicephalobus spp. eggs present in faeces were identified by molecular methods. Polymerase Chain Reaction (PCR) was used to amplify the partial 5′ variable region (~ 390 base pairs) of 18 S DNA using SSUA_F and SSU22_R primers. Furthermore, the PCR products obtained were sequenced, and phylogenetic analysis was performed using available sequences from GenBank. Results Microscopic examination of 141 fresh faecal samples revealed 5 fecal samples were infected with small ellipsoidal nematode eggs ranging between 40 and 50 × 50–60 μm. This study’s PCR amplicons showed ~ 390 bp bands on 2.0% agarose gel. A partial sequence of 18 S DNA (363 bp) was obtained herein (GenBank accession no. OQ843456). Conclusion Overall, using molecular tools represents a significant step forward in diagnosing and managing the Halicephalobus gingivalis infections in horses.

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