On 19th July 2007 re-occurrence of the H5N1 highly pathogenic avian influenza (HPAI) virus was noticed in Europe. The index strain of this novel H5N1 lineage was identified in the Czech Republic where it caused historically the first HPAI outbreak in commercial poultry. In the present study we performed molecular and phylogenetic analysis of the index strain of the re-emerging H5N1 virus lineage along with the Czech and the Slovak H5N1 strains collected in 2006 and established the evolutionary relationships to additional viruses circulated in Europe in 2005-2006. Our analysis revealed that the Czech and the Slovak H5N1 viruses collected during 2006 were separated into two sub-clades 2.2.1 and 2.2.2, which predominated in Europe during 2005-2006. On the contrary the newly emerged H5N1 viruses belonged to a clearly distinguishable sub-clade 2.2.3. Within the sub-clade 2.2.3 the Czech H5N1 strains showed the closest relationships to the simultaneously circulated viruses from Germany, Romania and Russia (Krasnodar) in 2007 and were further clustered with the viruses from Afghanistan and Mongolia circulated in 2006. The origin of the Czech 2007 H5N1 HPAI strains was also discussed.