ABSTRACTPreviously we demonstrated profound effects of dopamine transporter (DAT) SLC6A3 genotype on limbic responses to smoking cues (SCs). Probands carrying at least one copy of the 9‐repeat allele (9‐repeat carriers) had greater neural responses to SCs in the anatomically interconnected rostral ventral striatum/medial orbitofrontal cortex (VS/mOFC), compared with homozygotes for the 10‐repeat allele (10/10‐repeats). To test the reliability of the initial findings, we examined perfusion functional magnetic resonance images acquired during SC exposure in a new cohort of smokers (N = 26) who were genotyped for the SLC6A3 polymorphism. In smokers overall, activity was enhanced in the VS/mOFC (t = 3.77). Contrasts between allelic groups revealed that 9‐repeat carriers had a greater response to SCs in the VS (t = 3.12) and mOFC (t = 3.19). In separate groups, 9‐repeat carriers showed increased activity in the VS (t = 5.47) and mOFC (T = 4.96), while no increases were observed in 10‐repeats. Subjective reports of craving correlated with increased activity in reward‐related structures including the extended amygdala, insula and post‐central gyrus, and decreased activity in the dorsolateral prefrontal cortex, and were DAT‐genotype dependent (r = 0.63–0.96). In secondary analyses, we found that The Fagerström Test for Nicotine Dependence scores correlated with enhanced SC‐induced perfusion in 10/10‐repeats in the insula, mOFC, medial temporal and superior frontal gyri (r = 0.50–0.82), while correlations were absent in 9‐repeat carriers. Despite heterogeneity introduced by a host of factors, including variance in other genes involved in smoking behavior, we confirm that DAT genotype predicts the direction and location of neural responses to SCs.