Despite increasing evidence that large northern lakes are rapidly changing due to climate change, descriptive baseline studies of their physicochemical properties are largely lacking, limiting our ability to detect or predict change. This study represents a comprehensive scientific assessment of the limnology of Yukon’s largest lake: Lhù’ààn Mân’ (Kluane Lake), an important waterbody for local and First Nation communities, and key habitat for trout and salmon. Water sample and instrument data generated throughout 2015 describe distinct regions within the lake and their respective seasonal variability. A deep, glacially-influenced southern basin was characterized by cold, turbid, poorly stratified, unproductive, and nutrient-poor conditions; a shallower northwestern region (Tthe Kaala Daagur (Brooks/Little Arm)) was warmer, fully mixed, and more productive; a northeast region (’Ùha K’ènji (Talbot/Big Arm)) was clear and stratified with intermediate depth, temperature, productivity, and nutrient concentrations; and a central region had intermediate physicochemical conditions relative to the other three. This variability demonstrates the need for adequate spatial (within lake) and temporal (between seasons) monitoring of large northern lakes. In 2016, glacier recession within the watershed resulted in diversion of the lake’s primary inflow (‘A’ą̈y Chù’ (Slims River)). Our results, when used together with Indigenous knowledge, form a historical reference that enables assessments of the potential ecological consequences to Lhù’ààn Mân’.