Diatoms are the most species-rich group of microalgae, and their contribution to marine primary production is important on a global scale. Diatoms can form dense blooms through rapid asexual reproduction; mutations acquired and propagated during blooms likely provide the genetic, and thus phenotypic, variability upon which natural selection may act. Positive selection was tested using genome and transcriptome-wide pair-wise comparisons of homologs in three genera of diatoms (Pseudo-nitzschia, Ditylum, and Thalassiosira) that represent decreasing phylogenetic distances. The signal of positive selection was greatest between two strains of Thalassiosira pseudonana. Further testing among seven strains of T. pseudonana yielded 809 candidate genes of positive selection, which are 7% of the protein-coding genes. Orphan genes and genes encoding protein-binding domains and transcriptional regulators were enriched within the set of positively selected genes relative to the genome as a whole. Positively selected genes were linked to the potential selective pressures of nutrient limitation and sea surface temperature based on analysis of gene expression profiles and identification of positively selected genes in subsets of strains from locations with similar environmental conditions. The identification of positively selected genes presents an opportunity to test new hypotheses in natural populations and the laboratory that integrate selected genotypes in T. pseudonana with their associated phenotypes and selective forces.