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Premise of the study: Two marker systems were developed for the molecular identifi cation of three Trapa species based on the length variation of nuclear AP2 and trnL-F chloroplast intergenic spacer region. • Methods and Results: Our marker systems analyzed 118 individuals among 36 populations from three Trapa species. Trapa incisa was found to have a unique genotype from the other two species. Individuals of T. bispinosa were distinguished from T. japonica because the former had only a single AP2 genotype. Moreover, our results suggest a hybrid status of T. japonica in terms of two bands within an individual. One band was specifi c to T. japonica and the other one shared with T. bispinosa . • Conclusions: Our marker system demonstrates that nucleotide sequence variations can serve as a fast, reliable, and reproducible tool for molecular genotyping and examining the natural hybrid of water chestnut species. [ABSTRACT FROM AUTHOR]