Abstract Background: CYP I A1 polymorphisms, which have been reported to be associated with an elevated risk of lung cancer, are usually detected through conventional methods such as PCR-restriction fragment length polymorphism, allele-specific PCR and single-strand conformational polymorphism. Methods: An assay that makes use of differences in thermal stability between perfect match and non-perfect match hybrids has been developed. Two oligonucleotides probes for each CYP1A1 polymorphic site were designed and labeled with digoxigenin. After hybridization with amplified DNA fragment, the hybrids were detected with a colorimetric method and the genotype were identified by calculating the ratio of signals obtained with two probes. Results: The ratios for three genotypes obtained from 50 samples can be divided into three distinctive nonoverlapped groups when applying to m 1 and m 2 sites of CYP1A1 locus, which demonstrated the feasibility of this assay to detect CYP I A1 polymorphisms. Conclusion: Compared with other methods, this assay has lower cost, is fast, simple and is suitable for a screening test in routine laboratory.