BACKGROUND: Bladder Cancer (BCa) is the tenth most incidental malignancy worldwide. BCa is mostly attributed to environmental exposure and lifestyle, particularly tobacco smoking. The Aryl Hydrocarbon Receptor Repressor (AhRR) participates in the induction of many enzymes involved in metabolizing carcinogens, including tobacco smoke components. Additionally, studies have shown that smoking demethylates the (AhRR) gene in blood, suggesting AhRR demethylation as a specific serum smoking biomarker. OBJECTIVE: This study aimed to validate AhRR demethylation as a smoking biomarker in the target tissue and investigate its contribution to bladder carcinogenesis. METHODS: AhRR percent methylation was tested for its association with patient smoking status and oncogenic outcome indicators, particularly p53, RB1, and FGFR3 activating mutations, muscle-invasiveness, and tumor grade, in 180 BCa tissue-based DNA. RESULTS: Results showed significantly higher AhRR percent methylation in muscle-invasive compared to non-muscle invasive tumors (42.86% vs. 33.98%; p= 0.011), while lower AhRR methylation was significantly associated with FGFR3 Codon 248 mutant genotype compared to wild-type (28.11% ± 9.44 vs. 37.87% ± 22.53; p= 0.036). All other tested associations were non-statistically significant. CONCLUSIONS: Although AhRR methylation did not predict smoking status in BCa tumors, it seems to play a role in carcinogenesis and disease progression. Our findings make a basis for further research.