BACKGROUND: Allopolyploidy is a genomic structure wherein two or more sets of chromosomes derived from divergent parental species coexist within an organism. It is a prevalent genomic configuration in plants, as an important source of genetic variation, and also frequently confers environmental adaptability and increased crop productivity. We previously reported the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to be a promising host for biofuel production and that its genome is allopolyploid, which had never previously been reported in eukaryotic microalgae. However, the study of allopolyploidy in F. solaris was hindered by the difficulty in classifying the homoeologous genes based on their progenitor origins, owing to the shortage of diatom genomic references. RESULTS: In this study, the allopolyploid genome of F. solaris was tentatively classified into two pseudo-parental subgenomes using sequence analysis based on GC content and codon frequency in each homoeologous gene pair. This approach clearly separated the genome into two distinct fractions, subgenome Fso_h and Fso_l, which also showed the potency of codon usage analysis to differentiate the allopolyploid subgenome. Subsequent homoeolog expression bias analysis revealed that, although both subgenomes appear to contribute to global transcription, there were subgenomic preferences in approximately 61% of homoeologous gene pairs, and the majority of these genes showed continuous bias towards a specific subgenome during lipid accumulation. Additional promoter analysis indicated the possibility of promoter motifs involved in biased transcription of homoeologous genes. Among these subgenomic preferences, genes involved in lipid metabolic pathways showed interesting patterns in that biosynthetic and degradative pathways showed opposite subgenomic preferences, suggesting the possibility that the oleaginous characteristics of F. solaris derived from one of its progenitors. CONCLUSIONS: We report the detailed genomic structure and expression patterns in the allopolyploid eukaryotic microalga F. solaris. The allele-specific patterns reported may contribute to the oleaginous characteristics of F. solaris and also suggest the robust oleaginous characteristics of one of its progenitors. Our data reveal novel aspects of allopolyploidy in a diatom that is not only important for evolutionary studies but may also be advantageous for biofuel production in microalgae.