Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches in vitro. PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment (doxorubicin, carboplatin and meloxicam) were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.