The spatio-temporal distribution pattern of malaria in Yunnan Province, China was studied using a geographic information system technique. Both descriptive and temporal scan statistics revealed seasonal fluctuation in malaria incidences in Yunnan Province with only one peak during 1995-2000, and two apparent peaks from 2001 to 2005. Spatial autocorrelation analysis indicated that malaria incidence was not randomly distributed in the province. Further analysis using spatial scan statistics discovered that the high risk areas were mainly clustered at the bordering areas with Myanmar and Laos, and in Yuanjiang River Basin. There were obvious associations between Plasmodium vivax and Plasmodoium falciparum malaria incidences and climatic factors with a clear 1-month lagged effect, especially in cluster areas. All these could provide information on where and when malaria prevention and control measures would be applied. These findings imply that countermeasures should target high risk areas at suitable times, when climatic factors facilitate the transmission of malaria.