Presented in part: Africa Influenza Scientific Symposium, NCID, Johannesburg, South Africa, 7–11 December 2009; Options for the Control of Influenza, Hong Kong, SAR, 3–7 September 2010. ; BACKGROUND: 2009 Pandemic Influenza A(H1N1) was first detected in June 2009 in South Africa and later resulted in extensive transmission throughout Africa. Established routine surveillance programmes and collaboration between private and public sector laboratories allowed for comprehensive molecular epidemiological and antigenic investigation of the first and second waves of 2009 Pandemic Influenza A(H1N1) in South Africa. METHODS: We screened 9792 and 6915 specimens from patients with Influenza like illness (ILI) or severe acute respiratory infection (SARI) symptoms from surveillance programmes in hospitalised or outpatients in South Africa in 2009 and 2010, respectively for Influenza by RTPCR. Influenza-positive specimens were subjected to genetic and antigenic characterisation. Bayesian and Maximum likelihood analyses of the Hemaglutinin (HA)genes of 96 2009 Pandemic Influenza A(H1N1) strains were used for molecular epidemiological investigations. Hemaglutination inhibition assays and sequencing of the PB2 and neuraminidase genes were used to investigate pathogenicity and resistance mutations. RESULTS: The 2009 Pandemic Influenza A(H1N1) epidemic occurred as a second epidemic peak following seasonal H3N2 cases in 2009 and in 2010. Progressive drift away from the A/California/7/2009 vaccine strain was observed at both the nucleotide and amino acid level with 2010 strains clustering separate to 2009 strains. A few unique clusters of amino acid changes in severe cases were identified, but most strains were antigenically similar to the vaccine strain and no resistance or known pathogenicity mutations were detected. CONCLUSION: Despite limited drift observed over the 2 seasons in South Africa, circulating 2009 Pandemic Influenza A(H1N1) strains remained antigenically similar to strains identified in other Northern and Southern hemisphere countries from 2010 and 2011. ; Centers for Disease Control and Prevention, Atlanta, Georgia, USA. ; http://www.journals.uchicago.edu/toc/jid/current ; hb2013 ; ay2013