Oleaginous yeasts have emerged as promising microbial cell factories for lipid production, offering sustainable alternatives to traditional sources of biodiesel and nutraceuticals. In this study, the lipid accumulation potential of yeast strains isolated from two freshwater aquatic ecosystems in Cali, Colombia, was evaluated to identify novel candidates for biotechnological applications. A total of 56 strains were tested for their oleaginous nature using a gravimetric lipid assay with glucose as a carbon source. Of the assessed strains, 46.15% exceeded 20% lipid yields relative to the dry biomass. Seven strains were selected using glycerol as a carbon source, but only five yeasts were further characterized for their lipid profiles. Molecular identification revealed diverse species, including Aureobasidium sp., Papiliotrema rajashtanensis, Rhodotorula spp., and Clavispora lusitaniae. The selected strains demonstrated unique lipid profiles, with high proportions of monounsaturated and polyunsaturated fatty acids, such as oleic acid (C18:1) and linoleic acid (C18:2). In particular, Aureobasidium sp. accumulated uncommon fatty acids such as petroselinic acid under conditions induced by glycerol. This fatty acid, which has a double bond in position 6,7 and a melting point of 33 °C, highlights its potential as an alternative to margarine production, as well as a precursor to sophorolipids, estolide esters, soaps, and plastics. Rhodotorula sp. exhibited very long-chain fatty acids such as docosadienoic and docosatrienoic acids in its lipid profile. These findings underscore the biotechnological value of yeasts from lentic aquatic systems as sustainable lipid producers, paving the way for innovations in biofuels, nutraceuticals, and oleochemicals.