The genomes of many viruses mimic their hosts’ compositional biases in terms of nucleotide, dinucleotide, codon and codon pair usage. We have previously highlighted the importance of CpG and UpA dinucleotides in the influenza A virus (IAV) genome by demonstrating that reversal of the natural suppression of these dinucleotides results in significant viral attenuation. In order to assess the influence of other compositional biases in a tractable and unbiased system, we are utilising a library of 195 synonymous permutations of the GFP coding sequence that exhibit wide variation in genetic composition. To achieve expression of GFP-encoding transcripts by the IAV polymerase, three different reporter viruses were designed with GFP cloned either in place of the majority of the haemagglutinin (HA) coding sequence or cloned for co-linear expression either upstream of nucleoprotein (NP) or between non-structural 1 (NS1) and nuclear export protein (NEP). These GFP-sequence-containing transcripts mimic IAV genomic RNAs in their ability to act as templates for viral polymerase, both in polymerase reconstitution assays and in the production of GFP-encoding viruses. The reporter virus designs produced striking differences in GFP expression levels. Moreover, the magnitude of expression differences among the synonymous GFP variants varied substantially across the different viral systems and between infections in mammalian and avian cells. These results highlight the importance using the optimal reporter virus design for the system under study, exemplified through our investigations of how gene expression is affected by nucleotide usage.