Previous hematologic and serum biochemistry reference interval (RI) values have been established for donkeys in various geographic regions, life-stages, or for specific donkey breeds. The last extensive investigation establishing RIs for adult donkeys in the United States (U.S.) was published over three decades ago. We aimed to establish updated robust RIs using a reference population of apparently healthy adult donkeys from across the U.S. Standard sized (n = 102), miniature (n = 17), and mammoth (n = 1) donkeys from four different states were enrolled, with 20% of the study population including donkeys captured directly from the wild in Death Valley National Park, CA. RIs were established in accordance with the American Society for Veterinary Clinical Pathology and Clinical Laboratory Standards Institute guidelines. The findings will assist practitioners with the interpretation of their complete blood count and biochemistry panel results in U.S. donkeys. This study also highlights a comparison of results for some important analytes in U.S. donkeys compared to U.S. horses and previously established donkey RIs.