Diatoms are microalgae, known to present several characteristics that make them excellent bioindicators. In this study, we analyzed the epiphytic diatom communities of ponds, with the aim of exploring the genetic variability explained by physical, chemical or geographical variables. We selected 77 lakes within the Spanish part of the Duero River Basin characterized by wide chemical variability. In each lake, physical parameters were measured in situ and water samples were taken to determine nutrient levels in the laboratory. Diatom communities were collected and identified by DNA-metabarcoding, amplifying a genetic barcode of rbcL gene. We analyzed the phylogenetic signal for several environmental parameters at the genus level for the three most representative genera of these lakes. Our results indicate significant phylogenetic signals that differ from a genus to another. Processes related to spatial scales appear to be the main predictors controlling the genetic diversity of diatom communities in these ponds, with genetic variants differing among geographical areas for a given species. These molecular studies confirm the enormous diversity present in the diatom communities that inhabit these ecosystems, but further research is needed to apply diatom-based techniques in ponds monitoring or conservation programs. Our results reinforce the view that considering not only morphological but also molecular and ecological characters are necessary to describe new living diatom species, whenever possible, especially given the high number of species described each year