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Currently canine fibrosarcomas and peripheral nerve sheath tumours (PNSTs) are differentiated by their histopathological phenotype. Preliminary global transcriptomic analysis has identified genes with significant differential expression in both tumour types that may act as potential tumour markers. The aim of the present study was to establish reverse transcriptase polymerase chain reaction (RT-PCR) assays for the differentiation of formalin-fixed and paraffin wax-embedded tumours of both types. Fifty histologically well-defined examples of canine fibrosarcomas and PNSTs were characterized immunohistochemically for the expression of S100, laminin and PGP 9.5. RT-PCR assays for the potential fibrosarcoma markers FHL2-Ex4 and FHL2-Ex9 and the PNST markers GLI1 and CLEC3B were established and tested for their specificity and sensitivity to differentiate fibrosarcomas and PNSTs by their mRNA expression. Immunohistochemical analysis challenged the value of S100, laminin and PGP 9.5 for the diagnosis of PNSTs, since both PNSTs and fibrosarcomas showed similar expression of these proteins. In contrast, a combination of the markers GLI1 and CLEC3B differentiated PNSTs from fibrosarcomas with a sensitivity of 89% and a specificity of 87%. The proposed fibrosarcoma markers FHL2-Ex4 and FHL2-Ex9 failed to separate PNSTs and fibrosarcomas (sensitivity 50%, specificity 88%). The failure of these markers to unequivocally separate fibrosarcomas and PNSTs raises questions as to whether histologically uniform PNSTs are less uniform at the molecular level than expected or if both tumour types, despite their different morphology, are more closely related in terms of their histogenesis than previously thought.