Quantifying diet is essential for understanding the functional role of species in regard to energy processing, transfer, and storage within ecosystems. Recently, variance structure in the stable isotope composition of consumer tissues has been touted as a robust tool for quantifying trophic niche width, a task that has previously proven difficult due to bias in direct dietary analyses and difficulties in integrating diet composition over time. We use stable isotopes (C and N) to examine trophic niche width of two sympatric aquatic snakes, banded watersnakes (Nerodia fasciata) and black swamp snakes (Seminatrix pygaea) inhabiting a wetland where seasonal migrations of amphibian prey cause dramatic shifts in resource availability. Specifically, we characterize snake and prey isotope compositions through time, space, and ontogeny. We find that prey cluster into functional groups based on similarity in isotopic composition and seasonal availability. Overall, isotope variance structure suggests that Nerodia exhibit broader (more generalist) trophic niche width relative to Seminatrix. Moreover, Nerodia exhibit seasonal variation in isotope composition, suggesting seasonal diet shifts that reflect amphibian prey availability. Conversely, Seminatrix exhibit little seasonal variation but display strong ontogenetic shifts in δ¹⁵N and δ¹³C that closely parallel ontogenetic shifts in their primary prey, paedomorphic mole salamanders (Ambystoma talpoideum). Although niche dimensions are often viewed as static, our results demonstrate that seasonal shifts in niche width and position occur in Nerodia, leading to seasonal variation in niche overlap between the two snake species. Such short-term fluctuations in niche overlap have implications for our understanding of competitive interactions and consequently the structuring of communities and ecosystems.