AbstractMolecular methods to understand host feeding patterns of arthropod vectors are critical to assess exposure risk to vector‐borne disease and unveil complex ecological interactions. We build on our prior work discovering the utility of PCR‐Sanger sequencing blood meal analysis that work well for soft ticks (Acari: Argasidae), unlike for hard ticks (Acari: Ixodidae), thanks to their unique physiology that retains prior blood meals for years. Here, we apply blood meal metabarcoding using amplicon deep sequencing to identify multiple host species in individual Ornithodoros turicata soft ticks collected from two natural areas in Texas, United States. Of 788 collected O. turicata, 394 were evaluated for blood meal source via metabarcoding, revealing 27 different vertebrate hosts (17 mammals, five birds, one reptile, and four amphibians) fed upon by 274 soft ticks. Information on multiple hosts was derived from 167 individual O. turicata (61%). Metabarcoding revealed mixed vertebrate blood meals in O. turicata while same specimens yielded only one vertebrate species using Sanger sequencing. These data reveal wide host range of O. turicata and demonstrate the value of blood meal metabarcoding for understanding the ecology for known and potential tick‐borne pathogens circulating among humans, domestic animals, and wildlife such as relapsing fever caused by Borrelia turicatae. Our results also document evidence of prior feeding on wild pig from an off‐host soft tick for the first time in North America; a critical observation in the context of enzootic transmission of African swine fever virus if it were introduced to the US. This research enhances our understanding of vector‐host associations and offers a promising perspective for biodiversity monitoring and disease control strategies.