Larvae of Gasterophilus spp. (Diptera: Oestridae) cause gastrointestinal myiasis of equids. However, their identification may be problematic due to morphological similarities between species infesting identical regions of the digestive tract. In this study, genes encoding for mitochondrial cytochrome oxidase I (COI) and for the 16S and 28S ribosomal subunits of the most commonly encountered Gasterophilinae subfamily species [i.e., Gasterophilus haemorrhoidalis (L.), Gasterophilus inermis (Brauer), Gasterophilus intestinalis (De Geer), Gasterophilus nasalis (L.), and Gasterophilus pecorum (F.)] were studied, together with Gyrostigma pavesii (Corti), a rhinoceros parasite, and Hypoderma lineatum (De Villers), as outgroup taxa. Analysis identified interspecific differences that allowed their unequivocal identification. The high genetic homology among the sequences of G. haemorrhoidalis and G. intestinalis (i.e., 100, 99.86, and 99.46% in the 28S, COI, and 16S genes, respectively) strongly support the hypothesis that they are morphotypes of the same species. Phylogenetic analyses (maximum-likelihood and parsimony) were performed using PAUP; all analyses supported monophyly of subfamily Gasterophilinae. This study confirms the utility of the COI and 16S and 28S rRNA genes to address diagnostic and phylogenetic questions in Gasterophilus species.