The objective of this study was to explore the environmental factors having the greatest influence on the distribution and abundance of epipelic diatom species in different wetlands in southern Spain. We previously defined four groups of conductivity categories: fresh (< 0.8 mS cm−1), oligosaline (< 8 mS cm−1), mesosaline (8–30 mS cm−1) and eusaline (> 30 mS cm−1). A dbRDA analysis performed on a subset of 36 of the 53 wetlands, using a total of 25 environmental variables, showed that five environmental variables (conductivity, pH, wetland area, silicates, and total suspended solids) were the best explanatory variables for the diatom assemblage, with conductivity being the main explanatory variable. Nonmetric multidimensional scaling (nMDS) analysis performed on the set of 53 wetlands revealed significant differences in diatom composition among the four conductivity groups. The key species in the eusaline group were Tryblionella pararostrata, Halamphora sp.1 and Cocconeis euglypta, whereas in the mesosaline and oligosaline group, these were Navicula veneta, Tryblionella hungarica and Nitzschia inconspicua. Finally, in the fresh group dominated Achnanthidium minutissimum, Navicula veneta and Gomphonema exilissimum. This study on epipelic diatoms can therefore contribute to the knowledge of these organisms in a European region with a high diversity of wetland typologies.