Human carboxylesterases 1 and 2 (CES1 and CES2) catalyze the hydrolysis of many exogenous compounds. Alterations in carboxylesterase sequences could lead to variability in both the inactivation of drugs and the activation of prodrugs. We resequenced CES1 and CES2 in multiple populations (n = 120) to identify single-nucleotide polymorphisms and confirmed the novel SNPs in healthy European and African individuals (n = 190). Sixteen SNPs were found in CES1 (1 per 300 bp) and 11 in CES2 (1 per 630 bp) in at least one population. Allele frequencies and estimated haplotype frequencies varied significantly between African and European populations. No association between SNPs in CES1 or CES2 was found with respect to RNA expression in normal colonic mucosa; however, an intronic SNP (IVS10-88) in CES2 was associated with reduced CES2 mRNA expression in colorectal tumors. Functional analysis of the novel polymorphisms described in this study is now warranted to identify putative roles in drug metabolism.