Microscale conditions within metaphyton mats affected the distribution of diatom genera. We investigated the conditions within layers of floating filamentous algal mats and changes in diatom genera over 57 days using microprobes every 2, 4, and 6 cm down through mats held in floating nets. Mats were then collected, frozen, and sliced into 2 cm layers for analysis. Filamentous algae and their diatom epiphytes were identified, counted, and analyzed for nitrogen, phosphorus, carbon, silicon, ash-free dry mass (AFDM), and chlorophyll a. Light intensity, temperature, dissolved oxygen, dry mass, and AFDM all fell significantly with increasing depth in the mat. Diatom coverage per filament was calculated as an epiphyte area index (EAI) and was significantly higher at the edge versus the center of the mat. The uppermost 2 cm layer showed the greatest downward trend in EAI over the sampling period. The densities of Gomphonema, Cocconeis, and Fragilaria were significantly positively correlated with lower light intensity and lower layers of the mat. Cymbella/Encyonema density was significantly correlated with higher light intensity. Gomphonema, Cocconeis, and Nitzschia were positively correlated with filaments with higher chlorophyll a content. Achnanthidium, Cymbella/Encyonema, and Nitzschia required higher levels of silicon. Diatoms with different growth habits responded similarly to measured variables. Stalk-forming Gomphonema and adnate Cocconeis both occurred in lower light areas and grew well under low nitrogen and phosphorus conditions.