Two hundred eighty-two taxa of diatoms (Bacillariophyta) were identified from composited benthic samples collected at 16 sites on the Little River in western Kentucky in 2000 and 2003. The Little River basin is heavily impacted by non-point source pollution consisting of high nutrient inputs and siltation from agricultural and urban runoff. Pennate diatoms dominated the flora comprising >96% of the total taxa throughout the basin. Commonly occurring pennate species included Achnanthidium minutissimum, Amphora perpusilla, Cocconeis placentula var. euglypta, Gomphonema parvulum, Navicula cryptocephala, N. cryptotenella, N. menisculus, N. minima, N. seminulum, N. tripunctata, Nitzschia amphibia, N. dissipata, N. frustulum, N. palea, Planothidium lanceolata, and Sellophora seminulum. Most common centric species were Cyclotella meneghiniana and Melosira varians. The number of taxa found in the Little River was similar to other eastern North American streams and typical of streams impacted by agricultural and urban non-point source pollution (organic and nutrient enrichment and high siltation). A species checklist of all diatom taxa identified in the Little River with currently accepted nomenclature is presented as a baseline for future comparisons.