Diatom assemblage composition and water chemistry at four springs of different geological origin in western Kentucky and Tennessee (Land-Between-the-Lakes National Recreation Area, LBL) were examined using unglazed quarry tiles as artificial substrates. Significant differences in diatom species composition and water chemistry were found among the four springs. A total of 25 taxa were identified with 9 taxa being common to all springs. Calciphilous Planothidium lanceolata var. lanceolata (Bréb.) Grunow and Cocconeis placentula var. lineata (Ehr.) V. H. were the dominant species in the carbonate streams of limestone geology (Mint and Panther springs). Acidophilous Eunotia intermedia (Krasske ex. Hust), Achnanthidium minutissimum var. saprophila Kobaysi and Mayama, and Fragilariforma virescens (Ralfs) Williams and Round were most abundant in the streams with siliceous and argillaceous geology (Barnett and Brown Springs). Statistical analyses indicated that diatom species composition was most highly correlated with conductivity and suggested that the underlying geology of the springs played a role in periphyton community composition.