This study examines the diversity and distributional patterns of benthic diatomassemblages in the upland streams of Tamiraparani River system, southern part of Western Ghats ofPeninsular India. A total of 168 benthic diatoms representing 16 orders, 32 families, and 47 generawere enumerated. Structuring of diatom community was dependent on the environmental conditionsof the respective habitats. Canonical correspondence analysis described both physical habitat qualityand water parameter gradient and factors that correlated significantly were substrate type, watertemperature, dissolved oxygen and nitrate content. Gomphonema gandhii, Cocconeis placentula andNavicula cuspidata were strongly associated with sites with embedded substrates and clear water.Diatoms could be effective in assessing physical habitat alterations in streams.