Avian influenza caused infection and spread throughout Nigeria in 2006. Carcass samples (lung, liver, spleen, heart, trachea and intestine) from the different regions of Nigeria were processed for virus isolation. Infective allantoic fluids were tested for avian influenza viruses (AIV) and Newcastle disease virus using monospecific antisera. Thirty-five isolates were generated and characterized molecularly using the haemagglutinin gene. The molecular analysis indicated that different sublineages of the highly pathogenic avian influenza (HPAI) H5N1 viruses spread throughout Nigeria. We compared the Nigerian isolates with others from Africa and results indicated close similarities between isolates from West Africa and Sudan. Some of the analysed viruses showed genetic drift, and the implications of these for future epidemiology and ecology of avian influenza in Africa require further evaluation. The spread of primary outbreaks was strongly linked to trade (legal and illegal), live bird markets, inappropriate disposal, and poorly implemented control measures. No strong correlation existed between wild birds and HPAI H5N1 in Nigeria.