The present study investigated genetic variability within a population of Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) affecting horses in an endemic area of central Italy using polymerase chain reaction (PCR)-coupled sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). No different cox1 sequences were detected in any of the H. muscae individual, while two haplotypes representing H. microstoma individuals differed for one substitution. The pairwise distance between the H. muscae and H. microstoma was 11%, coding for five amino acid changes. The sequence of an informative region within the cox1 gene of H. microstoma and H. muscae was analyzed by Maximum Likelihood and Bayesian phylogenetic methods using available mitochondrial sequences spirurid taxa belonging to Filarioidea, Thelazioidea, and Habronematoidea. Phylogenetic analysis supported the split of the tree into two sister spirurid groups, Habronematoidea and Filarioidea + Thelazioidea. The phylogenetic and evolutionary implications of Habronema with Filaroidea and Thelazioidea are discussed.