Water chestnut (Trapa natans L.) is a group of annual, floating-leaved aquatic plants that serves as food and medical resources in many countries. However, the molecular method for distinguishing different T. natans L. resources is lacking. In this study, we detected genetic diversity of several chloroplast and nuclear genic or intergenic sequences in four varieties of T. natans and one wild type of Trapa incisa Siebold & Zuccarini to evaluate their potential as molecular markers. Our data revealed that the three chloroplast fragments (rbcL, matK, and pbsA-trnH) show no sequence difference among all tested samples. Only one nucleotide substitution is detected for the nuclear ribosomal internal transcribed spacer (ITS) in the T. natans variety Shuihongling. Four nucleotide substitutions are detected for the nuclear carotenoid isomerase (CRTISO) gene in the variety Hongxiuxie. In contrast, a total of 29 polymorphic sites are detected for a Toll and interleukin-1 receptor-nucleotide binding site–leucine rich repeat (TNL) gene in the five samples, among which six are nucleotide substitutions and the rest are insertions/deletions. The five samples could be fully distinguished from each other based on the TNL gene. To specifically authenticate ‘Heshangling’, 33 randomly amplified polymorphic DNA (RAPD) markers were adopted to amplify genomic sequences from the five samples. A pair of sequence characterized amplified region (SCAR) primers were designed based on the results of RAPD markers, which could specifically amplify one target band from all eight individuals of ‘Heshangling’, but none from any individuals of other T. natans varieties or one T. incisa. Taken together, a TNL sequence was provided in this study to distinguish four T. natans varieties and one T. incisa. Furthermore, a RAPD-SCAR marker was developed for efficient authentication of ‘Heshangling’.