We found mean target strength to be a reliable in situ predictor of fish weight, which allows direct estimation of the pelagic planktivore fish biomass from target strength measurements. Fish were collected by midwater trawling concurrent with target strength measurements (120-kHz frequency) in Lake Michigan. The mean weight of fish caught ranged from 2 to 71 g and mean target strength ranged from –54.9 to –38.0 decibels. Changes in mean target strength explained 73% of the variability in mean weight for combinations of various planktivore species, principally rainbow smelt Osrnerus mordax, bloaters Coregonus hovi, and alewives Alosa pseudoharengus. Bloaters were found to be less acoustically reflective than the other pelagic species, and a linear regression model with a classification variable was used to predict weight from target strength for bloaters and for the other species. We demonstrated that variations in the backscattering properties of different fish species must be considered to obtain accurate acoustic-based estimates of fish biomass.