Human carboxylesterase 1 (hCES1), encoded by the CES1 gene, is the predominant hepatic hydrolase responsible for the metabolism of many therapeutic agents, toxins, and endogenous substances. Genetic variants of CES1 can affect hCES1 function and expression and ultimately influence clinical response to drugs serving as hCES1 substrates. The CES1 gene consists of three isoforms including the functional CES1A1 and CES1A2 genes and the nonfunctional pseudogene CES1A3. Natural variants of these isoforms exert differing impacts on hCES1 function. However, the existing CES1 genotyping methods are incapable of determining whether these variants belong to CES1A1, CES1A2, or CES1A3 because of the high similarity among these three genes, as a consequence they are unable to discriminate between heterozygotes and homozygotes. We report the development of a novel long-range PCR-based, discriminative genotyping assay capable of specifically detecting the variants among CES1A1, CES1A2, and CES1A3 genes. The comparison of the genotyping results between this novel assay and those previously reported methods highlighted the necessity of applying the discriminative genotyping assay in pharmacogenetic studies involving CES1 gene.