Abstract Background Commercially available D-dimer assays use antibodies against human D-dimer, with limited sensitivity and specificity data in companion animals. Objectives To evaluate the immunoreactivity of D-dimer in plasma of dogs, horses, and cats with commercially available antibodies to human D-dimer. Animals Plasma samples were collected from healthy dogs and horses, and from surplus feline plasma submitted for diagnostic purposes. Methods Descriptive 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. Results The 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 Importance The 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.