African horse sickness virus (AHSV) is a ten-segmented, dsRNA virus, classified as a distinct species within the genus Orbivirus, family Reoviridae. There are nine serotypes of AHSV, any of which can cause African horse sickness (AHS), an extremely severe ‘transboundary’ and notifiable disease of horses, listed by OIE. AHS is currently restricted to sub-Saharan Africa, but has occasionally emerged causing major outbreaks in other geographic regions. Complete genome nucleotide sequences were determined for nine reference-strains of AHSV (different serotypes). The selection-pressure on each genome-segment and its encoded proteins, in relation to protein function were analysed in phylogenetic comparisons. This initial AHSV sequence database also provided a basis for molecular-epidemiology studies. In particular, the role of the Seg-2 in determination of virus-serotype was used to identify viruses involved in a multi-serotype outbreak, identifying Ethiopia as an important area of AHSV circulation. Phylogenetic-relationships were also investigated between different isolates of AHSV serotypes 2, 4, 6, 8 and 9, from various locations in Africa. Two real-time RT-PCR assays were developed for detection of the highly conserved genome segments 1 and 3, and molecular diagnosis of AHSV. Real-time RT-PCR assays were also developed, targeting Seg-2, for detection and identification of the nine AHSV serotypes. These assays are suitable for ‘high throughput’ characterisation of AHSV outbreak-strains, from either endemic or AHSV-free zones. The results obtained would facilitate rapid design and implementation of appropriate virus control measures. Cross-contamination was detected in four of the original AHSV reference-strains. A new set of plaque-cloned and monotypic reference-strains was therefore identified and characterised, and is available through the Orbivirus Reference Collection (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-Nos.htm). Serological relationships were analysed using antibodies against VP2 of AHSV-9 (expressed by recombinant MVA) and the nine monotypic reference-strains, showing neutralisation of both AHSV-9 and AHSV-6, in agreement with their closer phylogenetic relationship within Seg-2/VP2.