Vertical distribution of fish was studied immediately upstream of the Três Marias hydroelectric power plant dam from December 2010 to December 2011. Eighteen field trips were conducted for hydroacoustic sampling over a period of 24 h each time, with 6-h diel sampling intervals within each of the 18 sample dates. Gill nets were used to collect information on fish species composition and length. The greater fish abundance near the surface during the night and at higher depths during the daytime over a 1-day period suggests that the behavioural phenomenon known as diel vertical migration occurs at Três Marias reservoir. Therefore, the aim of the present study was to evaluate the occurrence of diel vertical migration of fish in a Neotropical reservoir, the possible relationship with the operation of the hydroelectric power plant and the variables that may affect fish distribution in water column. Analysis of some limnological and operational variables showed that water transparency, temperature and dissolved oxygen were correlated with vertical fish distribution patterns. These results are important for proposing management measures to mitigate the effects of hydroelectric power plants on fish, such as fish passage through turbines.