Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms. © 2004 Elsevier Inc. All rights reserved.