The nuclear genome is crucial for cells, and increasing evidence shows that nuclear DNA content alone can alter cell and organismal characteristics. However, the extent of genome size variation and its ecological and evolutionary consequences are not well understood, especially in microorganisms. We used flow cytometry to estimate genome size and GC content for 53 evolutionary lineages of the microalgal genus Synura (Chrysophyceae, Stramenopiles). Genome size evolution was reconstructed in a phylogenetic framework using molecular markers. A set of genomic, morphological, and ecogeographic variables characterizing Synura lineages were evaluated and tested as predictors of genome size variation in phylogeny-corrected statistical models. Both genome size and GC content varied widely in Synura, ranging from 0.19 to 3.70 pg DNA and from 34.0% to 49.3%, respectively. Genome size variation was mainly associated with cell size, less with the size of the silica scales covering the cell surface, and not at all with the phylogenetically conserved ultrastructure of the silica scales. The ecological requirements of Synura lineages were significantly affected by genome size variation, suggesting larger genomes are associated with habitats with higher soil nitrogen content, higher latitudes, and lower mean temperatures. The evolution of genome size in Synura suggests potential dynamism. Unlike genome size decreases, increases were mostly restricted to short terminal branches, indicating lower macroevolutionary stability. Lineages with larger genomes exhibited a narrower range of suitable ecological conditions, likely due to evolutionary constraints on upper genome size limits.