AbstractClinical response of clozapine is closely associated with serum concentration. Although tobacco smoking is the key environmental factor underlying interindividual variability in clozapine metabolism, recent genome‐wide studies suggest that CYP1A and NFIB genetic variants may also be of significant importance, but their quantitative impact is unclear. We investigated the effects of the rs2472297 C>T (CYP1A) and rs28379954 T>C (NFIB) polymorphisms on serum concentrations in smokers and nonsmokers. The study retrospectively included 526 patients with known smoking habits (63.7% smokers) from a therapeutic drug monitoring service in Norway. Clozapine dose‐adjusted concentrations (C/D) and patient proportions with subtherapeutic levels (<1070 nmol/L) were compared between CYP1A/NFIB variant allele carriers and homozygous wild‐type carriers (noncarriers), in both smokers and nonsmokers. Clozapine C/D was reduced in patients carrying CYP1A‐T and NFIB‐C variants versus noncarriers, both among smokers (−48%; p < 0.0001) and nonsmokers (−35%; p = 0.028). Patients who smoke carrying CYP1A‐T and NFIB‐C variants had a 66% reduction in clozapine C/D versus nonsmoking noncarriers (p < 0.0001). The patient proportion with subtherapeutic levels was 2.9‐fold higher in patients who smoke carrying NFIB‐C and CYP1A‐T variants versus nonsmoking noncarriers (p < 0.0001). In conclusion, CYP1A and NFIB variants have significant and additive impact on clozapine dose requirements for reaching target serum concentrations. Patients who smoke carrying the studied CYP1A and NFIB variants, comprising 2.5% of the study population, may need threefold higher doses to prevent risk of clozapine undertreatment. The results suggest that pre‐emptive genotyping of NFIB and CYP1A may be utilized to guide clozapine dosing and improve clinical outcomes in patients with treatment‐resistant schizophrenia.