Previously 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 to 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 FTND 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.