Abstract The Neotropical region has a wide aquatic biodiversity, which is affected by anthropogenic activities. Mining has caused a negative impact on these ecosystems, directly affecting benthic communities. Aquatic macroinvertebrates are effective bioindicators of water quality, especially Chironomidae larvae, since these show high species richness, abundance, and sensitivity to human activities. The genus Polypedilum is among the most representative of the family Chironomidae. Research on these species is focused on their abundance and dominance in water bodies. In addition, several studies have addressed alterations in the external morphology of some Chironomidae due to mining pollution (heavy metals). However, little is known about the effects of mining on the internal morphology of these species. This study provides the first histological description of different systems and organs of Polypedilum sp. larvae. Furthermore, we report histopathological alterations in larvae collected from two tributaries affected by mining, namely Toldafría and La Elvira streams located in the municipalities of Villamaría and Manizales (Caldas – Colombia). Our findings show target organs of mining pollution in a species of Polypedilum, which represent potential early-warning histopathological biomarkers with relevant implications for water quality monitoring.