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Simple Summary: Humans have interacted with reptile and amphibian species for millennia. The current study was designed to collect knowledge about the use of amphibian and reptile species by the native peoples residing along the Jhelum and Chenab rivers in Punjab, Pakistan. To the best of our knowledge, this is the first quantitative assessment of the cultural uses of amphibian and reptile species in the study area. However, hunting, trade, and cultural use are the greatest threats to the diversity of the amphibians and reptiles in the studied area. These threats can potentially lead to their extinction. It is important to protect the highly endangered and vulnerable species employed in therapeutic medications, more specifically in terms of their conservation. Amphibians and reptiles have interacted with humans for millennia. However, humans interact with amphibian and reptile species in different manners, which depend on their culture and traditions. This study was designed to better understand the interactions between amphibian and reptile species and their usage among the native peoples in the vicinity of the Jhelum and Chenab rivers, Pakistan. Information was collected through semi-structured interviews and questionnaires, and was analyzed by using different indices, including the frequency of citation, corrected fidelity level, fidelity level, relative importance level, and informant major ailment. Two amphibians and twenty-six reptile species were used in therapeutic medicine in the study area. Based on the cultural analysis, we found that Naja naja (black cobra) was highly cited across all cultural groups. A 100% Fidelity Level was calculated for the following species: Naja naja (eye infection), Varanus bengalensis (joint pain), Eurylepis taeniolatus (cataract), and Acanthodactylus cantoris (cancer). We found five endangered species in the study area, i.e., Aspideretes gangeticus, A. hurum, Chitra indica, Varanus flavescens, and Geoclemys hamiltonii, that were used to cure joint pain, muscle stretching and pain, backbone pain, paralysis, and psoriasis, respectively. Likewise, Lissemys punctata andersoni, a vulnerable species as labelled by the International Union for Conservation of Nature, was extensively used for the treatment of joint pain, body pain, paralysis, and arthritis in the study area. In terms of conservation, it is critical to protect the highly vulnerable and endangered species that are being used in therapeutic medicines. Our findings may be helpful for the conservation of amphibian and reptile species by helping to make an effective plan to prevent their extinction. The main threats to the diversity of amphibian and reptile species in the area are hunting, trading, and cultural use. These threats could potentially lead to the extinction of these species. Therefore, with the involvement of concerned authorities, e.g., local stakeholders, the Ministry of Climate Change, provincial wildlife departments, academia, and conservation managers, immediate conservation measures should be taken for the protection and sustainable utilization of medicinal species. [ABSTRACT FROM AUTHOR]