Background <p>Dental pain is influenced by both local inflammatory processes and neurobiological and psychological factors. This study compared substance P (SP) and calcitonin gene-related peptide (CGRP) levels in pulp tissue and unstimulated saliva between patients with dental pain and painless controls, and investigated their relationships with pain severity, pulpal inflammation, and psychological distress.</p> Methods <p>Fifty-two adult patients indicated for primary endodontic treatment were enrolled and allocated into two groups: 26 patients with dental pain and 26 painless controls with no pain complaint and a VRS score of 0. Pain intensity was assessed using the Verbal Rating Scale (VRS). Depression, anxiety, and stress were evaluated using the Depression Anxiety Stress Scale-21 (DASS-21). Pulp tissue and unstimulated saliva samples were collected and analyzed using enzyme-linked immunosorbent assay. Pulpal inflammation was graded histopathologically. Between-group comparisons were performed using independent samples t-tests or Mann–Whitney U tests, as appropriate. Effect sizes were calculated using eta squared (η²). Correlations were assessed using Pearson or Spearman coefficients to examine associations between neuropeptide levels, pain severity, and inflammation grade.</p> Results <p>Pulp calcitonin gene-related peptide levels were significantly higher in patients with dental pain than in painless controls (<i>p</i> = 0.007; η² = 0.146). Salivary substance P levels were also significantly increased in the painful group (<i>p</i> = 0.044; η² = 0.080). No significant between-group differences were observed for pulp substance P or salivary calcitonin gene-related peptide (<i>p</i> &gt; 0.05). Depression, anxiety, and stress scores were all significantly higher in patients with dental pain (all <i>p</i> &lt; 0.001). Pain severity showed positive correlations with pulp calcitonin gene-related peptide (<i>r</i> = 0.332) and salivary substance P (<i>r</i> = 0.324), whereas no significant association was found between pain severity and histopathological inflammation grade.</p> Conclusions <p>Pulp calcitonin gene-related peptide may be associated with local neurogenic activation in symptomatic pulpal pain, while salivary substance P may represent a potential noninvasive candidate marker for pain-associated responses. However, the lack of consistent pulp–saliva correspondence indicates that salivary biomarkers should not be considered direct surrogates of pulpal neuropeptide activity.</p> Trial registration <p>ClinicalTrials.gov Identifier: NCT07330024.</p> Graphical abstract <p></p>