To gain further insight into the molecular epidemiology of Hantaan virus (HTNV) in Guizhou, China, rodents were captured in this region in 2004 and 2005. In addition, serum samples were collected from four patients. Ten hantaviruses were isolated successfully in cell culture from four humans, two Apodemus agrarius, three Rattus norvegicus and one Rattus nitidus. The nucleoticle sequences for their small (S), medium (M) and partial large (L) segments were determined. Phylogenetic analysis of the S and M segment sequences revealed that all of these isolates belong to the species HTNV, suggesting a spillover of HTNV from A. agrariLIS to Rattus rats. All available isolates from Guizhou were divided into four distinct groups either in the S segment tree or in the M segment tree. The clustering pattern of these isolates in the S segment tree was not in agreement with that in the M or L Segment tree, showing that genetic reassortment between HTNV had occurred naturally. Analysis of the S segment sequences from available HTNV strains indicated that they formed three clacles. The first clacle, which comprised only viruses from Guizhou, was the outgroup of clacles 11 and 111. The viruses in the second clacle were found in Guizhou and mainly in the far-east Asian region, including China. However, the viruses in the third clacle were found in most areas of China, including Guizhou, in which haemorrhagic fever with renal syndrome (HFRS) is endemic. Our results reveal that the highest genetic diversity of HTNV is in a limited geographical region of Guizhou, and suggest that Guizhou might be a radiation centre of the present form of HTNV.