Ostreococcus, exhibit remarkable ecological success despite possessing small and compact genomes. Here we investigate how these picoeukaryotes maintain adaptive potential within severe genomic constraints, focusing on a unique feature of Mamiellales genomes: two outlier chromosomes containing extensive low-GC regions with distinct genetic characteristics. Using a multi-omics approach combining Hi-C, transcriptomics, genome-wide methylation profiling, and metabolomics, we examined the physical and regulatory insulation of these outlier chromosomes in Ostreococcus and related species.