This study was designed to define the potential clinical relevance of identifying alterations affecting p53 pathway in bladder cancer and to test a new, low-cost, high-throughput, and array-based TP53 sequencing technology. Tumor samples from 140 evaluable patients with bladder cancer were analyzed with two methods to detect TP53 gene mutations, including single-stranded conformational polymorphism followed by direct sequencing and an oligonucleotide array-based sequencing method. Immunohistochemistry was used to assess patterns of expression of p53, p21/WAF1, and mdm2. Median follow-up time was 27.6 months. Results from the above analyses were correlated with clinicopathological parameters and outcome. Combining the mutation-detection assays, 79 cases (56.4%) were found to harbor TP53 gene mutations. Direct sequencing identified 66 point mutations and five frameshift mutations. The p53 oligonucleotide array detected 65 point mutations and four splice site mutations in different exons but missed all five frameshift mutations. p53 nuclear overexpression was observed in 71 cases (50.7%), lack of p21 nuclear expression was found in 81 cases (57.9%), and mdm2 nuclear overexpression was seen in 64 cases (45.7%). In multivariate analysis, 17 patients (12.1%) had an altered p53 pathway, defined by the detection of mutant TP53 and/or p53 nuclear overexpression, loss of p21 nuclear expression, and mdm2 nuclear overexpression, and exhibited the worst clinical outcome in the observation period (P = 0.015), and it appears to be a significant prognostic factor associated with patient survival.