Strongylus vulgaris ranks among the most dangerous parasitic nematodes affecting horses worldwide. This large strongyle invades both gastrointestinal and vascular tissues, leading to verminous arteritis, severe colic episodes, and considerable mortality in affected animals. Its continued presence in domestic horse populations poses substantial challenges for veterinary practitioners, especially in geographic areas where equine agriculture remains economically important. The present investigation was designed to determine the prevalence of Strongylus vulgaris among equine populations in Al-Najaf Province, Iraq. We employed both conventional microscopy and molecular techniques for parasite detection. Furthermore, phylogenetic relationships were examined, and potential associations with host gender, age category, and collection season were evaluated. Between October 2025 and February 2026, we obtained 200 fresh fecal specimens from horses of varying ages and both sexes across multiple locations in Al-Najaf Province. Initial screening involved direct smear examination and flotation procedures under light microscopy. Subsequently, 100 samples underwent molecular analysis through polymerase chain reaction (PCR) targeting the ITS1 gene, which yielded an amplification product of 290 base pairs. Phylogenetic reconstruction was accomplished using MEGA11 software package. Microscopic analysis revealed an overall infection rate of 34.5% (69 out of 200 samples). Male horses exhibited a slightly elevated infection frequency (38.75%) relative to females (31.67%), though this difference lacked statistical significance (P > 0.05). Adult horses demonstrated significantly higher infection rates (42.0%) compared with younger foals (27.0%) (P ≤ 0.05). Seasonal variation showed peak prevalence during December (45.0%) and lowest rates in November (20.0%). Molecular confirmation through PCR identified S. vulgaris in 30% of tested samples (30/100) with high significance (P < 0.001). Five local sequences were successfully deposited in NCBI GenBank under accession numbers (PZ012358, PZ012359, PZ012360, PZ012361, and PZ012362.