Choline acetyltransferase is critical in the synthesis of acetylcholine and regulation of cholinergic neuron functions. We recently reported association of the encoding gene ChAT with both smoking cessation and nicotine dependence (ND) in two independent European American (EA) samples; however, in the replication sample, only limited SNPs partially covering the gene were examined. In this study, we examined the association of 14 SNPs, which cover the entire gene, with ND, assessed by smoking quantity (SQ), heaviness of smoking index (HSI), and Fagerström Test for ND (FTND), in 2,037 subjects from 602 families of African American (AA) or EA origin. Individual SNP-based association analysis revealed that five SNPs showed nominal association with at least one ND measure in one of the samples (P = 0.022-0.042); none remained significant after correction for multiple testing. Haplotype-based association analysis revealed that haplotypes G-G-A-C, formed by rs1880676-rs3810950-rs10082479-rs8178990 (P = 0.005-0.0178), and G-G-T-C-G-C, formed by rs1880676-rs3810950-rs10082479-rs8178990-rs3793790-rs12266458 (P = 0.00247-0.00468), displayed significant association with all three ND measures in the AA sample, as did haplotype T-C-G-A-T, formed by rs12266458-rs11101191-rs8178991-rs4838544-rs4838547 (P = 0.00741-0.0103), in the EA sample. All these detected haplotype-based associations remained significant after correction for all major haplotypes for a given SNP combination. Together, our findings, in conjunction with the previous report of the association, warrant further investigation of ChAT in ND.