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For the last two decades, a rapid eutrophication process impacts Lake Titicaca, the largest tropical freshwater lake in South America and the main highest Great Lake. This is especially notorious in the Bolivian sector of its shallow Lago Menor sub-basin. Lago Menor is deteriorated by the combination of multiple contaminations (domestic, industrial and mining) from untreated wastewater discharged from the urban area of El Alto, indiscriminate overfishing, and climate change. These threats particularly affect the native Andean killifish genus Orestias, the ecology and dynamics of which require in-depth studies with non-invasive techniques. Here, we use hydroacoustic method to evaluate fish patterns of distribution and abundance in the Lago Menor. Hydroacoustic data were collected during the 2015 rainy season (November) with vertical beaming at 120 kHz along transects that sum a total length of 140 km. Our results showed that the proxy of fish biomass was linked to Lago Menor bathymetry. Furthermore, the vertical fish biomass proxy was steady from 3 to 20 m. This pilot study provides the first image of fish, mostly Orestias spp., distribution and opens future studies to deepen knowledge on their ecology and ethology, and regular monitoring of their population and stock for the fisheries assessment in Lake Titicaca.