Background: Different interpretations of ayurvedic texts have recognized/recommended taxonomically distinct species/varieties of Trichosanthes as “Patola,” which is used extensively in the Indian traditional system of medicine for curing a variety of disorders. Sources of “Patola” also vary depending on region-specific availability and diverse traditions in different parts of India, which creates ambiguity in correct identity of “Patola.” Since “Patola” is one of the medicinal plant species traded in high volumes, its exact taxonomic identity and authentication is very essential. Objectives: Molecular profiling of different taxa recognized as “Patola” was carried out for correct identification and authentication of commercial crude drug samples. Materials and Methods: Molecular marker profiles of different taxa recognized as “Patola” were generated using inter-simple sequence repeat (ISSR) markers. Attempt was also made to authenticate commercial crude drug samples, using ISSR markers. Results: Selected 10 polymorphic ISSR primers generated distinct amplification profiles for the taxa traded as “Patola.” Moreover, two varieties of Trichosanthes cucumerina (var. cucumerina and var. anguina) could be clearly distinguished based on the specific bands produced by primers UBC 812, 822, 887, 888, and 889. Molecular profiling of market samples along with the genuine plant samples confirmed the trade of different Trichosanthes species as well as Momordica charantia under the name of “Patola.” Conclusion: To our knowledge, this is a first report on the application of ISSR markers for identification and authentication of “Patola.” Developed DNA profiles should be useful in investigations of adulteration in market samples and establishment of correct identity.