The Suquía River middle-lower basin (Córdoba, Argentina) is subjected to a strong anthropogenic impact because it receives pollutants from different sources. The main goal of this study was to evaluate the introduced fish species Gambusia affinis as a bioindicator of environmental pollution in the middle-lower basin of the Suquía River. The assessment was performed by measuring biomarkers at different levels of biological organization, at two sampling sites (before and after Córdoba city), and at dry and wet seasons. Water quality evaluation was made through a water-quality index (based on physico-chemical parameters), heavy-metals and pesticides concentrations in water. The water quality varied between sampling sites, showing the most degraded conditions downstream Córdoba city. The same pattern of variation was detected in the biomarkers studied, mainly in: gill and liver histopathological indexes, copulatory organ (gonopodium) morphology and vitellogenin expression in males and females. The present study characterized the environmental conditions in the middle-lower basin of the Suquía River and revealed the low freshwater quality at the most polluted site. Although G. affinis is an introduced species, it could be considered a good sentinel of water resource quality of invaded Neotropical basins. Our results demonstrated the importance of addressing the environmental quality monitoring through an integrated analysis of water quality parameters together with histological, morphological and molecular parameters. Thus, our study provides a good model for application in other basins of South America.