Phytoplankton has an essential role as a primary producer in lakes, providing information on environmental conditions and water quality. The objective of this research was to inventory, characterize, and assess the diversity of microalgae in the Quimsacocha lake system in Azuay, Ecuador. Water samples were collected from the two most important Quimsacocha lakes to morphologically characterize the microalgae and evaluate their diversity. The diversity and physicochemical parameters were measured to evaluate and explain differences in community richness and composition using multivariate analysis (NMDS and PERMANOVA). The level of organic pollution in the lakes was estimated using the Pollution Index, and the trophic status was assessed using dominant phytoplankton (AARL-PP Score). Eighty morphospecies were identified within 25 genera, 16 families and 8 classes. The Bacillariophyceae class was the most diverse, represented by five families. The genera Synedra, Chlorella, and Cosmarium were the most abundant, suggesting a moderate level of organic pollution and a mesotrophic state in the lakes. Physicochemical parameters such as temperature, pH, dissolved oxygen and electrical conductivity were similar in both lakes. Nonetheless, the lake depth was different, varying between 0.9 and 8 m in LQA (with one extreme value of 22 m), compared to LQB, which had depths between 7 and 19 m. Alpha diversity revealed a significant difference (p = 0.0001) in species abundance between the two lakes, while specific richness was similar (p = 0.8213). In terms of beta diversity, a significant difference in species composition was observed (p = 0.001). This study provides base-line information regarding the ecological status of these Andean lakes, being of great help in developing conservation plans to preserve their biodiversity and water quality for human consumption.