Lymphoma is the most common hematopoietic tumour in dogs and has significant overlap with the human disease. Tumour biomarker discovery is providing new tools for diagnosis and prediction of response to therapy and prognosis. MicroRNAs (miRNAs) are small non-coding RNAs that participate in post-transcriptional gene regulation and aberrant expression affects hallmarks of cancer. The aim of this study was to characterize miRNA expression from dogs with multicentric B or T cell lymphoma compared to healthy control dogs. We further compared expression between lymph nodes and corresponding plasma samples and assessed changes in expression at relapse compared to diagnosis. Lastly, we investigated miRNAs for association with clinical outcome in patients treated with CHOP chemotherapy. An initial pilot study was completed by profiling 277 canine miRNAs in lymph nodes to help design a custom PCR array with 38 targets. Quantification was performed using real time RT-PCR and relative expression was determined by the delta-delta Ct method. In the pilot study we identified altered expression (>5-fold) of 40 miRNAs for B cell lymphoma and 44 miRNAs for T cell lymphoma. The custom array was then used on a larger sample population (n=45) in a prospective study. In lymph nodes, there were 16 miRNAs with significantly altered expression for B cell lymphoma and 9 for T cell lymphoma. In plasma, there were 15 miRNAs altered for B cell lymphoma and 3 for T cell lymphoma. The majority of miRNAs did not have correlated expression between lymph node and plasma and only 8 miRNAs were significantly different between diagnosis and relapse. For B cell lymphoma, 8 miRNAs had differential expression in the non-remission group compared to dogs that completed CHOP in remission. Four of these miRNAs were also altered in patients that died prior to one-year. Kaplan-Meier survival curves for high versus low miRNA expression revealed that 10 miRNAs were correlated with progression-free survival and 3 with overall survival. This study highlights miRNAs of interest for canine multicentric lymphoma. Future goals include development of miRNA panels that may be useful as biomarkers with the intent to provide improved outcome prediction to veterinary cancer patients.