When using biomarkers such as n-alkanes as tools for paleo-environmental reconstructions, it is imperative to determine their specific sources for each setting. Towards that goal, we analysed a set of various potential organic matter (OM) sources such as aquatic and terrestrial plants, dust, and soils from Laguna Potrok Aike (LPA) and surrounding areas in Southern Patagonia. We determined chain length distributions and hydrogen (δD) and carbon (δ13C) isotopic compositions of n-alkanes of different OM sources in order to quantify their relative contributions to lake sediments. Our results reveal that mid-chain n-alkane, n-C23, is predominantly produced by submerged aquatic plants, whereas long-chain n-alkanes (n-C29 to n-C31) are derived from various terrestrial sources. We estimated their relative contributions to the sediment using two approaches, i.e. based on the n-alkane distributions and their δD and δ13C values. Both approaches result in similar estimates of aquatic and terrestrial contributions for mid- and long-chain n-alkanes to the sediment. 62-73% of the mid-chain n-C23 alkanes originate from aquatic sources while 66-77% of the long-chain n-alkanes originate from dust and 14-30% from terrestrial plants. Our study shows that mid-chain n-alkanes such as n-C23 alkane in LPA are derived mainly from aquatic macrophytes and thus have the potential to record changes in lake-water isotopic composition. In contrast, the n-C29 alkane reflects the isotopic signal of various terrestrial sources from Southern Patagonia.