PURPOSE: To investigate the stability of a novel type of markers used in partial bladder tumor irradiation and tumor deformation as indicated by the markers. MATERIALS AND METHODS: In 15 patients with solitary bladder cancer, lipiodol was injected in the bladder wall during flexible cystoscopy to identify the tumor. A planning CT scan was made, followed by daily cone-beam CT (CBCT) scans during treatment. To study the accuracy of using these markers for image guidance, uncertainties U1 and U2 were calculated, which were defined as the difference between submask registration (covering single marker) and the average of all submask registrations and the difference between the submask registration and the general mask registration (including all markers), respectively. Finally, to study tumor deformation, the relative movement of each marker pair was correlated with the relative bladder volume (RBV). RESULTS: The analyzed patients had 2.3 marker injections on average. The lipiodol spot size was 0.72 +/- 1.1 cm(3). The intensity of spots in both CT and CBCT was significantly higher than the surrounding bladder tissue. The uncertainties U1 and U2 were comparable, and the uncertainties in left-right direction (0.14-0.19 cm) were smaller than those in cranial-caudal and anterior-posterior directions (0.19-0.32 cm). The relative marker movement of within-zone marker pairs was much smaller (and has less dependence on the RBV) than across-zones marker pairs. CONCLUSIONS: Lipiodol markers are a feasible method to track bladder tumor by using online CBCT. Tumor deformation is observed, especially for tumors that cross the defined bladder zones.