CYP2D6 bioactivates codeine and tramadol, with intermediate and poor metabolizers (IMs and PMs) expected to have impaired analgesia. This pragmatic proof-of-concept trial tested the effects of CYP2D6-guided opioid prescribing on pain control. Participants with chronic pain (94% on an opioid) from seven clinics were enrolled into CYP2D6-guided (n = 235) or usual care (n = 135) arms using a cluster design. CYP2D6 phenotypes were assigned based on genotype and CYP2D6 inhibitor use, with recommendations for opioid prescribing made in the CYP2D6-guided arm. Pain was assessed at baseline and 3 months using PROMIS measures. On stepwise multiple linear regression, the primary outcome of composite pain intensity (composite of current pain and worst and average pain in the past week) among IM/PMs initially prescribed tramadol/codeine (n = 45) had greater improvement in the CYP2D6-guided versus usual care arm (-1.01 ± 1.59 vs. -0.40 ± 1.20; adj P = 0.016); 24% of CYP2D6-guided versus 0% of usual care participants reported ≥30% (clinically meaningful) reduction in the composite outcome. In contrast, among normal metabolizers prescribed tramadol or codeine at baseline, there was no difference in the change in composite pain intensity at 3 months between CYP2D6-guided (-0.61 ± 1.39) and usual care (-0.54 ± 1.69) groups (adj P = 0.540). These data support the potential benefits of CYP2D6-guided pain management.