Population-genomic analyses are essential to understanding factors shaping genomic variation and revealing lineage-specific sequence constraints and adaptation. The dearth of such analyses for unicellular eukaryotes prompted us to assess variation in Paramecium, one of the most well-studied ciliate genera. The aurelia complex consists of ~15 morphologically indistinguishable species that diverged subsequent to two rounds of whole-genome duplications (WGDs), perhaps as long as 320 MYA, and are well known for their streamlined genomes and extremely short introns. We examine patterns of polymorphism by sequencing whole genomes of 10-13 worldwide isolates of each of three species belonging to the Paramecium aurelia complex: P. tetraurelia, P. biaurelia, P. sexuarelia, and two outgroup species that do not share the WGDs: P. caudatum and P. multimicronucleatum. An apparent absence of strong global geographic population structure suggests continuous or recent dispersal of Paramecium over long distances. Introns and intergenic regions are highly constrained relative to 4-fold degenerate sites, more so in species with smaller intergenic regions. Nuclear genomic diversity in the intergenic regions is reduced for ~100-150 bp, both upstream and downstream of genes, suggesting the presence of densely packed regulatory modules. The beginning and end (~70-100 bp) of protein-coding genes are more constrained than the rest of the gene. Comparison of sequence variation at non-synonymous and synonymous sites allows identification of possible candidates of duplicate genes that might be undergoing non-functionalization and provides insights into differential evolutionary constraints on single-copy versus multi-copy genes. This study serves as a first attempt at a population-genomic analysis in Paramecium, and provides an excellent resource for future studies in evolutionary and functional genetics in ciliates.