Splenic masses are common in dogs and vary dramatically in their clinical behavior. Clinically, and even with histology, it can be challenging to differentiate between benign and malignant splenic masses. Hemangiosarcoma (HSA), the most common malignancy of the spleen, is a very aggressive tumor with a poor prognosis. We hypothesize that microRNAs (miRNAs) in mass tissue and serum can differentiate between HSA and other splenic masses. Fifty-nine miRNAs were investigated by reverse-transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR) in serum and/or tissue from dogs with HSAs (serum