Canine mammary sarcomas are usually very aggressive and easily metastasize. Unfortunately the biology of this type of tumor is not well known because they are a very rare type of tumors. The aim of this study was to find differences in gene expression patterns in canine mammary osteosarcomas (malignant) versus osteomas (benign) using DNA microarrays. Our microarray experiment showed that 11 genes were up-regulated in osteosarcoma in comparison to osteoma whereas 36 genes were down-regulated. Among the up-regulated genes were: PDK1, EXT1, and EIF4H which are involved in AKT/PI3K and GLI/Hedgehog pathways. These genes play an important role in cell biology (cancer cell proliferation) and may be essential in osteosarcoma formation and development. Analyzing the down-regulated genes, the most interesting seemed to be HSPB8 and SEPP1. HSPB8 is a small heat shock protein that plays an important role in cell cycle regulation, apoptosis, and breast carcinogenesis. Also SEPP1 may play a role in carcinogenesis, as its down-regulation may induce oxidative stress possibly resulting in carcinogenesis. The preliminary results of the present study indicate that the up-regulation of three genes EXT1, EIF4H, and PDK1 may play an essential role in osteosarcoma formation, development and proliferation. In our opinion the cross-talk between GLI/Hedgehog and PI3K/AKT pathways may be a key factor to increase tumor proliferation and malignancy.