The JWA gene is a novel cell differentiation-related gene thought to be a responsive gene in response to DNA damage and repair induced by environmental stressors. Recently, a novel single nucleotide polymorphism (SNP) was identified in the promoter of the JWA gene (-76GC) that may alter the transcription activity and thus play a role in increased risk of bladder cancer. Further, studies were conducted to screen for more novel variants in the JWA exons by using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) followed by PCR-RFLP (PCR restriction fragment length polymorphism) methods. Finally, the functional relevance of the newly identified genetic variants in a hospital-based case-control study of 215 bladder cancer patients and 250 cancer-free controls was evaluated. In addition to the -76GC polymorphism, another novel SNP (454CA in exon2 and 723TG in exon 3) of JWA was identified. The -76GC allele and genotype frequencies were found to vary in different ethnic groups. The -76C allele and 454A allele were both associated with significantly increased risk of bladder cancer. In contrast, the 723GG genotype was associated with a decreased risk of bladder cancer. Furthermore, -76C and 454A together increased the risk of bladder caner using haplotype and stratification analysis. In conclusion, the three novel functional genetic polymorphisms of JWA gene, -76GC, 454CA, and 723TG, appear to contribute to the etiology of bladder cancer.