West Nile Virus (WNV) infections resulting from the bite of an infected mosquito is of emerging concern for the health of people for all parts of the country including Kansas . The virus also infects various bird species, horses and other vertebrate animals. WNV was first reported in the US in New York in the year 1999 and then spread to many parts of the country. In this study, the prevalence of WNV in Kansas was evaluated by a modern molecular method that allows for accurate and rapid detection. The analysis was performed for the years 2001 to 2005 in mosquitoes, dead birds, and clinically suspected animals. WNV was detected in mosquitoes from the year 2002 to 2005. About 6%, 13.5%, 16.8%, and 8.7% of the total mosquito pools analyzed were tested positive for the years 2002, 2003, 2004, and 2005, respectively. Significantly higher percentages of dead birds were tested positive i.e., about 62%, 61%, and 47% for the years 2002, 2003, and 2004, respectively. WNV positives in Kansas included horses, mules, mountain goats, sheep, and squirrels. This is the first study demonstrating the establishment and increased spread of WNV in Kansas . Further, we discovered that the virus detected in Kansas is similar to that originally described in New York . The continued detection of WNV in mosquitoes at high percentages in Kansas is of significant public health concern and calls for continued surveillance and public health activities. (Funding: Kansas Department of Health and Environment.)