A male mixed breed dog with clinical Hemophilia B was tested with a direct-to-consumer dog genomics service. After being genotyped as “normal” at three previously published Hemophilia B variants, all coding regions of the dog’s F9 gene were sequenced. A single basepair deletion in exon 5 of F9 led to a frameshift mutation leading to a premature truncation of FIX, including loss of the catalytic domain. The dam for this individual is a Great Pyrenees suggesting this X chromosome variant could segregate within the Great Pyrenees and possibly related breeds. The discovery of this novel variant could be applicable to the Great Pyrenees community should hemophiliac Great Pyrenees be observed at appreciable frequency and emphasizes the utility of genetic testing in mixed breed dogs.