Cnesterodon decemmaculatus, which is a freshwater fish abundant in the Pampean region of Argentina, is used as a bioindicator of the status of watercourses throughout its distribution area. This work aimed to (1) evaluate the genetic diversity and population structure of C. decemmaculatus from two river basins in north-eastern Buenos Aires province, Argentina, (2) assess a possible association between genetic structure and the geographical distribution of river basins, including in the analysis data of populations from the south of the province, and (3) validate the use of the species as a bioindicator of the ecological status of watercourses in north-eastern Buenos Aires province. The analysis of a 700-bp fragment of the mitochondrial DNA control region showed moderate to high levels of genetic diversity and lack of genetic structure between the north-eastern basins, validating the use of this species as a bioindicator. Significant differences in genetic structure were found among basins at a regional scale. The lack of genetic structure between the north-eastern basins may result from historical and current events, whereas genetic differences between the north-eastern and southern basins may be explained by the absence of physical connections between them.