The aim of this study was to evaluate the presence of influenza virus co-infections in humans and changes in the genetic variability of A(H3N2) virus strains in southern Italy from 1999 to 2009. A partial sequence of the haemagglutinin (HA) gene by human influenza H3N2 strains identified in oropharyngeal swabs from patients with influenza-like illness was analysed by DNA sequencing and a phylogenetic analysis was performed. During the seasons 1999–2000, 2002–2003, 2004–2005 and 2008–2009, the influenza viruses circulating belonged to subtype H3N2. However, A(H1N1) subtype virus and B type were respectively prevalent during the 2000–2001, 2006–2007, 2007–2008 and 2001–2002, 2003–2004, 2005–2006 seasons. The HA sequences appeared to be closely related to the sequence of the influenza A vaccine strain. Only the 2002–2003 season was characterized by co-circulation of two viral lineages: A/New York/55/01(H3N2)-like virus of the previous season and A/Fujian/411/02(H3N2)-like virus, a new H3 variant. In this study, over the decade analysed, no significant change was seen in the sequences of the HA gene of H3 viruses isolated.