The sequence of the S10 gene segment of the United States prototype strains of BTV serotypes 10, 11, 13, and 17 obtained from the American Type Culture Collection (ATCC), the commercial modified live virus vaccine strains of BTV serotypes 10, 11, and 17, and 20 field isolates of BTV serotypes 10, 11, 13, and 17 was determined to better define the molecular epidemiology of BTV infection in the US. All S10 gene segments were 822 nucleotides in length with two in-frame initiation codons (nucleotides 20 to 22 and 59 to 61) and a single termination codon (nucleotides 707 to 709), thus all S10 genes were predicted to encode two proteins (NS3, NS3A). Nucleotide differences between the S10 genes from field isolates of BTV ranged from zero (100% identity) to 142 (81.8% identity). The sequences of the S10 gene segments from the US prototype ATCC strains of BTV 10 and 11 were very different from the previously published sequences of putative US prototype viruses of the same serotypes (Lee and Roy, 1986; Hwang et al., 1992). Comparison of the predicted NS3/NS3A proteins encoded by the S10 gene showed little variation between the various viruses (from 93 to 100% identity). This apparent conservation of NS3/NS3A amongst different strains and serotypes of BTV likely is a reflection of functional constraints on the protein that tolerate little variation. The various US isolates of BTV segregate into two distinct monophyletic groups based on their S10 gene sequences and clustering of viruses was independent of serotype, year of isolation, geographical origin, and of host species of isolation. The S10 sequence data also show that viruses that segregated within each of these two monophyletic groups co-circulated in the western US between 1953 and 1990, and that reassortment of the S10 gene segment likely occurs in nature. Comparison of dendograms derived from sequence analysis of the S3 (de Mattos et al., 1996)and the S10 gene segments from the same viruses also indicates that the S10 gene segment evolves and reassorts independently of the S3 gene segment.