Aquatic organisms exist in a constantly fluctuating habitat, with changes in photoperiod, temperature, pH, dissolved organic content, dissolved oxygen and food supply. Organisms must alter past of their physiological and biochemical processes to cope with these changes. We compared the effect of seasonal variations in factors related to energy metabolism of two species of sympatric amphipods, Hyalella pleoacuta and Hyalella castroi. The animals were collected monthly from April 2004 through March 2006. Contents of glycogen, proteins, lipids, triglycerides and the levels of lipoperoxidation were determined in males and females throughout the year by using spectrophotometric methods. Observations revealed significant seasonal differences in biochemical composition, as well as differences among sexes and species. Environmental conditions (e.g., trophic conditions) and reproduction are supposed to be the main processes influencing the seasonal patterns of variation in biochemical composition. Both species of Hyalella show ecological and behavioral differences, especially by resources such as food, space and seasonal variations of energy metabolism, which might facilitate their coexistence in the same habitat.