Studying the spatio-temporal variation in fatty acid signatures can increase our understanding of the nearshore food web structure as it continues to shift due to anthropogenic factors. The purpose of this experiment was to compare fatty acid signatures (FAS) of four fish species (alewife, round goby, spottail shiner, and yellow perch) from Lake Michigan. Fish were collected (n=300) along the southwestern shore of Lake Michigan during spring, summer, and fall of 2013 at three sampling sites with different habitat complexity. Their substrates were characterized as sand (site A), rocky (site B), and coarse sand with intermittent cobble and random boulders (site C). Fish were analyzed and FAS were determined based on the quantities of 29 different fatty acids. Significant differences in FAS among fish species were detected (ANOSIM, overall R = 0.796), with alewife and round goby presenting the most distinct FAS (25.5% dissimilarity). The fatty acids responsible for the most variation among species included 16:1n-7, 18:1n-9, 20:5n-3, and 22:6n-3. Spatial and temporal variations in FAS were observed within species. Fatty acid signatures of round goby collected at site B in spring and summer differed significantly (overall R 0.693 and 18.8% dissimilarity), which implies seasonal dietary shifts. Spatial differences in yellow perch FAS were also observed, indicating habitat driven plasticity in yellow perch diets. Although within species spatio-temporal FAS variations were observed, among species FAS differences were consistently larger. These data can be compared to samples from other years to determine annual changes as well. In addition, they can be compared with FAS of predatory fish to determine the diets of those species.