Purpose The aim of this prospective study was to evaluate the relationship between pain intensity and concentrations of salivary pain and stress biomarkers during orthodontic tooth movement. Methods Eighteen patients (8 males, 10 females; mean age 14.57 +/- 2.39 years) who needed orthodontic treatment with maxillary premolar extraction and segmental canine distalization participated in this study. Baseline samples were collected (T1), and orthodontic attachments were placed to maxillary first molars, second premolars, and canines. Then extractions were performed. After 1-month follow-up, canine distalization started with a segmental wire (T2). Concentrations of salivary alpha-amylase (sAA), cortisol, secretory immunoglobulin A (sIgA) and chromogranin A (CgA) were examined at T1, T2, and on days 4 (T3), 7 (T4), 14 (T5), and 30 (T6) after starting retraction. Participants also scored their pain on a visual analogue scale (VAS). Pain catastrophizing behavior and dental anxiety levels of the participants were evaluated by the Pain Catastrophizing Scale (PCS) and Corah's Dental Anxiety Scale (C-DAS), respectively. Repeated measure ANOVA, Mann-Whitney U test and Spearman's rank correlation coefficient analysis were used for statistical evaluations (p < 0.05). Results The maximum values for sAA were seen at T1. Males had higher sAA levels than females with statistical differences at T1, T3, and T4. No significant differences for cortisol, sIgA, and CgA concentrations were observed. The highest mean VAS score was recorded at T3. No correlations were detected between any salivary biomarkers, VAS, C-DAS, and PCS scores. Conclusions The stress of starting orthodontic treatment increased sAA levels more than the pain that was experienced during orthodontic tooth movement. Being male was a predictor of higher sAA concentrations. Orthodontic tooth movement did not cause significant alterations in salivary pain and stress biomarkers.