The main goal of the research was to investigate the influence of altitude and environmental factorson diatom periphyton communities composition in the Sava river from its source in Zelenci to Jesenice na Dolenjskem. We took winter (March 2015) and summer (July 2015) samples at nine locations. At each sampling site, we gathered 5–7 stones and sampled 42 cm2 periphyton (epilithon) altogether from the stones surfaces. Selected abiotic factors were measured, using the multi-meter. It was possible to observe that temporal and spatial parameters changed, especially temperature, oxygen saturation in water and conductivity. Chemical analyses were carried out in the laboratory and results showed that the content of nitrogen (nitrate, ammonium, total nitrogen) and phosphorus (orthophosphate, total phosphorus) is increasing downstream. In periphyton samples, 118 different species of diatoms were determined, with the highest number of algae species in the sample being 48 (sample KK2). The most abundant species were Achnanthidium minutissimum and Achnanthidium pyrenaicum. Cyclotella meneghiniana appeared in the samples downstream from the Šentjakob, what is untypical for the periphyton. The most diverse genera in the samples were Fragilaria, Navicula, Gomphonema, Nitzschia and Cymbella. The trophic and saprobic index values are increasing downwards and show that the river’s course becomes more congested by the distance from the source. The Shannon-Wiener (S-W) diversity index is higher in winter; however, it is increasing downstream in both seasons. The composition of the associations is primarily influenced by temperature and distance from the source. Associations which are closer to the source are more similar.