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Oral cancer is a major public health concern in South Asia, particularly in Pakistan, India, and Bangladesh, largely due to the widespread use of smokeless tobacco (SLT) products. The World Health Organization (WHO) classifies SLT as a Group 1 carcinogen. These products contain glycotoxins, such as methylglyoxal (MGO) that is well-known to cause genotoxicity by reacting with DNA. One of the most stable MGO-derived DNA adducts is N2-(1-carboxyethyl)-2′-deoxyguanosine (CEdG), which has been reported to cause DNA damage and genetic instability. However, the role of CEdG in the pathogenesis of oral cancer remains unstudied, and the MGO content in various SLT has not been previously determined. To our knowledge, this study represents one of the first investigations into the abundance of CEdG in oral cancer and its association with chromatin organization, while also quantifying MGO levels in SLT. CEdG was detected using a semi-quantitative in situ immunofluorescence assay and correlated with chromatin computational morphometry to evaluate CEdG-associated alterations in chromatin organization. Using ELISA, we also assessed MGO levels in various SLT, revealing elevated concentrations in commonly used products such as gutka, naswar, and khaini. Our results demonstrated significantly higher levels of CEdG in cancerous nuclei and were strongly correlated with disorganized chromatin texture. Together, these findings suggest that exposure to MGO-rich SLT is associated with CEdG accumulation and subsequent chromatin disorganization, which may be relevant to tobacco-associated oral carcinogenesis.