design: Feces, strongyle larvae from coproculture and luminal strongyle worms were collected from 12 naturally infected untreated equines at necropsy. Luminal worms were collected from three different gut compartments: caecum, ventral colon and dorsal colon. All equines were part of a research herd of the University of Kentucky, known as 'Population S', which is known to be infected with Strongylidae species resistant to benzimidazole and tetrahydropyrimidine drug classes. DNA was isolated using the NucleoSpin Soil kit (Macherey-Nagel, Dren, Germany) and the NucleoSpin Tissue XS kit (Macherey-Nagel, Dren, Germany) for luminal worm samples. The COI_Nema_Fw and COI_Nema_Rv primers (Duscher et al., 2015) with incorporated ILLUMINA adapter sequences were used in the first PCR. Indices were added in a second PCR using uniquie combinatios of P7 and P5 primers. The KAPA HiFi HotStart ReadyMix was used for all PCR amplifications. Final PCR products were purified using CleanNGS magnetic beads at a ratio of 10:8. DNA concentrations were quantified with a Qubit 4 fluorometer, and fragment quality was evaluated using the TapeStation system. All samples were sequenced on an Illumina MiSeq platform using the V3 600-cycle kit (2 300 bp). Following demultiplexing and quality filters, reads from both ends of each sample were paired using the justConcatenate mode of DADA2s mergePairs function. BLASTn (Altschul et al., 1990) was used to filter out ASVs that did not match at least 80% matching identity and 90% query coverage inside the Strongylidae family. The COI-database (Diekmann et al., 2025) was used to assign species.