Diatoms are unicellular organisms, they occur either as solitary cells or in colonies. Diatoms have two distinct shapes: a few centric diatoms and most pennate diatoms, they are surrounded by a cell wall made of silica. The present study tries to account for the diversity of Diatoms in Parambikulam Tiger Reserve, a protected area situated in the biodiversity hotspot of Western Ghats, India. The study was carried out over a period of one year, from October 2022 to September 2023 in three seasons (pre-monsoon, monsoon, and post-monsoon) from the freshwater resources within the sanctuary like rivers, reservoirs, and ponds. The surface water samples were collected in plastic containers with the help of a plankton net of mesh size 20µm, samples were fixed in 4% formalin for permanent preservation and detailed identification. Diatom specimens were photographed and identified based on relevant published literature. Bacillariophycean members are difficult to identify, so a clearing process is applied using concentrated nitric acid and permanent slides were prepared using naphrax mounting medium. Scanning electron microscopy is also done for morphology analysis. The current study identified a total of sixty-one taxa, which are distributed under 26 genera. Obtained genera were Achnanthidium (1), Amphora (3), Aulacoseira (1), Caloneis (2), Cocconeis (2), Cyclotella (1), Cymbella (3), Diploneis (1), Encyonema (3), Eunotia (3), Fragilaria (1), Gomphonema (4), Gyrosigma (1), Hantzchia (1), Luticola (1), Melosira (1), Navicula (10), Neidium (2), Nitzschia (9), Pinnularia (4), Planothidium (1), Pleurosigma (1), Rhopalodia (1), Surirella (2), Ulnaria (1), Stauroneis (1). The species Gyrosigma scalproides (Rabenhorst) Cleve and Nitschia irremissa Cholnocky are found to be new reports to Kerala. This is the first study available with regard to the diversity of diatoms in the Parambikulam Tiger Reserve.