Oxidative stress is involved in the pathogenesis of many conditions and is caused by free radicals in concentrations that overwhelm the natural scavenging mechanisms and cause pain and inflammation. This investigation sought to determine whether pain from temporomandibular disorders was associated with increased oxidative stress as measured by biomarkers in saliva and serum. Both salivary and serum levels of the oxidative stress biomarkers including 8-hydroxydeoxyguanosine, malondialdehyde and total antioxidant status were compared in patients with mild and severe TMJD pain and with healthy controls. These biomarkers were determined spectrophotometrically in saliva and serum from 10 high TMJD pain patients, 10 low TMJD pain patients, and 10 healthy control subjects from National Institute of Dental Research's TMJ Implant Registry and Repository. Linear and logistic regression analyses were used to evaluate the association between each biomarker and TMJD pain. The mean levels of log 8-hydroxydeoxyguanosine (saliva P < 0.0001, serum P = 0.0008), malondialdehyde (saliva P = 0.002, serum P = 0.004) and total antioxidant status (saliva P = 0.005; serum P = 0.001) achieved statistically significant differences between groups. In linear regression analysis, both salivary and serum levels of each biomarker were associated with TMJD pain. In a multivariable analysis, again, both salivary levels and serum levels were also different between groups. Salivary levels of oxidative stress ratios of 8-hydroxydeoxyguanosine, malondialdehyde and total antioxidant status were significantly different between patients with TMJD pain and controls and was comparable to that in serum. These biomarkers hold promise as a potential diagnostic and therapeutic strategy.