The genetic characteristics and phylogenetic relationships between the U.S. prototype strain of bluetongue virus serotype 10 (BTV 10), the modified live virus vaccine currently used in California, and three field isolates of BTV 10 obtained in 1980 and three in 1990 in California were determined by comparison of their L2 gene sequences. The L2 genes of the 1980 field isolates were very closely related to the L2 genes of the prototype strain and the vaccine strain, differing by only 0.1 to 0.5%. The 1990 field isolates diverged from all the other viruses by an average of 4.8%. They showed a high degree of genetic similarity that ranged from 98.2 to 99.7% and formed a separate group. All BTV 10 viruses derived from a common ancestor (bootstrap value 100%) from which two different lineages have diverged giving rise to two monophyletic groups, one including all the 1990 viruses and the other the prototype, the vaccine, and all 1980 field strains. The bootstrap analyses placed a 100% confidence value at each of these two nodes. These results indicate that two different lineages of BTV 10 circulated in California between 1953 and 1990. The effect of the vaccine on the evolutionary pathways of the BTV 10 population present in California in 1980 was not clearly established, but it did not influence the evolution of the BTV 10 field isolates obtained in 1990.