Intermittent rivers and ephemeral streams are critical yet understudied ecosystems, particularly in semi-arid regions where hydrologic variability governs community structure and ecosystem processes. This study utilized nine reaches across three hydrologic regimes (perennial and temporally stable flows, intermittent flows, and seasonally variable but perennial flows) along the San Saba River in central Texas (USA) to assess spatiotemporal responses of organismal diversity and food web structure across two seasonal periods (wet versus dry season). It is predicted that fish and macroinvertebrate diversity will be lower in intermittent reaches, whereas reaches with temporally stable and perennial flows will have the highest diversity. In addition, it is predicted that food chain length is greater in stable and perennial reaches. Study reaches were sampled twice (wet season and dry season) over the course of the study and water chemistry, benthic algae, terrestrial detritus, macroinvertebrates, and fish were sampled in both pool and riffle meso-habitats. Diversity (taxonomic richness) of macroinvertebrates and fish were assessed within and across mesohabitats, sites, and hydrologic regime types. Stable isotopes (δ¹³C and δ¹⁵N) of periphyton, macroinvertebrates, and fish were used to examine food chain length, basal resource dependance (allochthonous versus autochthonous resources), and the degree of niche diversity and overlap within and across mesohabitats, sites, and hydrologic regimes. Results indicate that fish taxonomic richness was greatest in perennial reaches and in pool mesohabitats, specifically. Macroinvertebrate community richness was also greater in perennial reaches, but the highest taxonomic richness occurred in riffle habitats. Preliminary results suggest that pool mesohabitats in perennially flowing reaches had greater food chain lengths and niche diversity in fishes.