BackgroundCommercially available D‐dimer assays use antibodies against human D‐dimer, with limited sensitivity and specificity data in companion animals.ObjectivesTo evaluate the immunoreactivity of D‐dimer in plasma of dogs, horses, and cats with commercially available antibodies to human D‐dimer.AnimalsPlasma samples were collected from healthy dogs and horses, and from surplus feline plasma submitted for diagnostic purposes.MethodsDescriptive research study. A cross‐linked fibrin lysate was prepared from plasma samples, and SDS‐PAGE and immunoblotting were performed with a variety of commercially available antibodies to human D‐dimer.ResultsThe selected antibodies demonstrated variable reactivity with D‐dimer of each species. The monoclonal antibody DD44 bound canine D‐dimer with good specificity and sensitivity, but this antibody did not react with feline or equine D‐dimer. The polyclonal antibody D2D bound putative D‐dimer in dogs, cats, and horses with good specificity, and higher sensitivity compared to human D‐dimer.Conclusions and Clinical ImportanceThe variable performance of commercially available human D‐dimer assays between species is, in part, because of inter‐species variation in D‐dimer immunoreactivity. The use of these assays should follow validation studies. Monoclonal antibody DD44 could be a focus for the development of a canine‐specific assay.