<p>Synonymous codon usage plays an important role in the adaptation of viruses to their hosts. Bovine viral diarrhea virus (BVDV) relies on a high mutation rate in its genome to achieve the necessary fitness in a particular host. However, the question of which selective forces influence nucleotide pair and synonymous codon usage patterns in different BVDV genotypes remains unresolved. Here, 169 BVDV strains isolated at different times in various countries were analyzed to compare their dinucleotide frequency and synonymous codon usage. Examination of the nucleotide usage pattern in the open reading frame (ORF) of BVDV revealed a significantly higher frequency of purine than pyrimidine, with the highest extent of nucleotide usage bias observed in the first codon position. Moreover, a nucleotide pair bias, especially favoring CpG dinucleotides, was observed in all of the genotypes. Together, the nucleotide composition constraints and nucleotide pair bias appear to have influenced the overall codon usage pattern. Nucleotide pair and synonymous codon usage biases were associated with individual genotypes to different degrees. Of particular note, BVDV-1 exhibited more variation in its nucleotide pair and synonymous codon usage than BVDV-2 and BVDV-3, suggesting that these patterns are shaped both by selection of mutations in the viral genome and translational selection in the host.</p>