BACKGROUND Biomarkers assayed from gingival crevicular fluid (GCF) are a potential tool for endodontic diagnosis and for monitoring treatment response. This cross-sectional study measured cytokines in GCF from teeth with apical periodontitis and evaluated their relationship with preoperative pain and other clinical findings. METHODS Participants presenting for root-end resection surgery due to apical periodontitis diagnosis (n = 56) underwent standardized clinical testing and completed preoperative questionnaires. GCF from diseased and control teeth were collected, processed, and analyzed. Mann-Whitney U and Wilcoxon tests were used to examine the cytokine levels in diseased compared to healthy control teeth. We also assessed the relationship of cytokine levels with clinical findings. RESULTS Interleukins (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ, and tumor necrosis factor-⍺ (TNF-⍺) were detected in GCF. TNF-⍺ levels were significantly higher in GCF collected from diseased versus control teeth (P = .02) and increased IL-1β levels in diseased teeth were detected (P = .06). Lower IL-10 levels were observed in teeth with a sinus tract and/or swelling compared to teeth without a sinus tract and/or swelling (P = .08). Cytokine levels did not clearly relate to the presence of pain. CONCLUSIONS Elevated levels of proinflammatory cytokines, including TNF-⍺ and IL1- β, were detected in GCF from diseased teeth compared to the healthy controls. Additional studies are needed to further investigate the utility of these biomarkers for objectively evaluating periradicular pathology.